Bent out of shape: α-Synuclein misfolding and the convergence of pathogenic pathways in Parkinson’s Disease

Abstract

Protein inclusions made up primarily of misfolded α-synuclein (α-Syn) are the hallmark of a set of disorders known as synucleionopathies, most notably Parkinson’s disease (PD). It is becoming increasingly appreciated that α-Syn misfolding can spread to anatomically connected regions in a prion-like manner. The protein aggregates that ensue are correlated with neurodegeneration in the various yet select neuronal populations that are affected. Recent advances have begun to shed light on the spreading and toxicity mechanisms that may be occurring in PD. Several key emerging themes are arising from this work suggesting that α-Syn mediated neurodegeneration is due to a combination of relative α-Syn expression level, connectivity to affected brain regions, and intrinsic vulnerability to pathology.

1. Introduction

Parkinson’s disease (PD) is the most common motor disorder and second most common neurodegenerative disorder after Alzheimer’s disease, affecting an estimated 7 million people worldwide [1, 2]. As age is a significant risk factor, PD prevalence is expected to rise sharply as the average life expectancy among the general population continues to increase [3] Despite its immense social and economic impact, our knowledge regarding the etiology of this condition remains incomplete and no effective treatments which modify the course of disease are presently available.

The most prominent feature in the brains of PD patients is the selective loss of dopaminergic (DA) pigmented neurons within the substantia nigra (SN) and to a lesser extent those residing in the ventral tegmental and retrorubral areas. Significant degeneration is also observed in other nuclei and neurotransmitter systems including the dorsal raphe (serotonergic), locus coeruleus (noradrenergic), nucleus basalis of Meynert (cholinergic) and the dorsal motor nucleus of the vagus nerve [4]. Degeneration in these areas likely contributes to both motor and non-motor symptoms observed in PD, especially hallucinations, depression, and sleep disorders.

For over a century, it has been recognized that neurodegenerative diseases are commonly associated with the accumulation of abnormally folded proteins within or in the vicinity of cells of the central nervous system (CNS) [5]. PD is characterized by the presence of eosinophilic inclusions in the soma of neurons termed Lewy bodies (or Lewy neurites when present in the neurites). Lewy bodies (LBs) represent a complex amalgamation of lipids, neuromelanin, and up to several hundred individual proteins with a key component being alpha-synuclein (α-Syn), an 140 amino-acid protein that is enriched in presynaptic vesicles of vertebrates [6–8]. A highly soluble lipid-binding protein normally thought to regulate synaptic vesicle release through stabilization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, α-Syn in Lewy pathology occurs as highly ordered amyloid-type fibrils [6, 9, 10]. In addition to abnormal conformations, α-Syn in PD brains is also commonly post-translationally modified by cleavage, hyperphosphorylation, ubiquitination, nitrosylation, and oxidation [11–16].

Several lines of evidence point to α-Syn playing a key, and possibly causal, role in PD. For example, the genetic studies that led to the discovery of α-Syn as a major component of LBs also revealed several point mutations which result in autosomal dominant PD [17–21]. Duplication or triplication of the wildtype locus that lead to 1.5–2 fold overexpression also give rise to dominantly-inherited PD, while several polymorphisms in the promoter region are reported to elevate the risk of developing disease [22–24].

The significance of α-Syn is further supported by large-scale genome-wide association studies that indicate a clear link between the SNCA locus and PD [25, 26]. Intriguingly, several strong genetic risk factors may have strong interactions with α-Syn. For example, mutations in glucocerebrosidase, which causes the lysosomal storage disorder Gaucher’s disease, also elevates α-Syn levels and increases the risk of PD by up to 30-fold in some populations [27, 28]. Moreover, Lewy pathology has also been reported in multiple forms of familial PD, such as those involving mutations in DJ-1, PINK1, and GBA, although this is less certain for other genetic forms of PD (e.g., LRRK2, Parkin, ATP13A2) [29–34]. Collectively, these data point to α-Syn dysfunction as a key process in PD pathogenesis. Indeed, the observation that cytoplasmic α-Syn inclusions are also characteristic of several other neurodegenerative conditions, most notably multiple system atrophy (MSA) and dementia with Lewy bodies, suggests that α-Syn accumulation is not merely a benign product of the degenerative process [5, 6, 35, 36].

Despite the overwhelming evidence for a central role of α-Syn in PD and other synucleinopathies and the explosion in our knowledge regarding this protein in both health and disease, two fundamental questions still remain unanswered. Firstly, what instigates the neurodegeneration that typifies PD? Secondly, why do selective cell populations undergo degeneration? This review discusses some of the recent advances that illustrate the complex pathobiological relationships between α-Syn misfolding and PD.

2. Prion-like properties of pathologic α-Syn

The distribution of α-Syn inclusions observed in the CNS of patients with PD and other synucleinopathies is highly non-uniform. On the contrary, Braak and colleagues have previously demonstrated that α-Syn inclusions in PD form in a predictable manner during disease progression, allowing categorization into at least six distinct clinicopathological stages in the majority of PD patients. Lewy pathology typically begins in the olfactory bulb and deep brain stem nuclei (stages I and II). This pattern correlates with several known prodromal symptoms of PD including olfactory impairment, autonomic dysfunction, and REM sleep disturbances [37]. Inclusion pathology is then detectable in more rostral regions, most notably midbrain DA neurons in stages III and IV. It is during these stages that the motor symptoms associated with PD become evident. In the ultimate stages (V and VI) pathology begins to be observed in the neocortex and has been correlated with the onset of dementia [38, 39]. While this stereotypic staging system appears to be valid in up to 80% in multiple patient subpopulations, other studies point to a significant portion of patients (up to 50%) that do not follow this classification [40–43].Heterogeneity in the initial location where α-Syn aggregates are first detected appears to be a source of this discrepancy resulting in modified or alternative staging systems that attempt to encompass populations that do not fit the conventional caudo-rostral pattern of spread, such as in dementia with LB [44]. Nonetheless, human studies clearly point to the gradual yet inevitable spread of α-Syn pathology originating from select regions. In conjunction with this evidence, post-mortem examination of fetal mesencephalic neurons transplanted into PD patients show that they develop Lewy pathology in a time-dependent manner. As fetal neurons initially lack any of the pathological processes and were unlikely to contain α-Syn inclusions at the time of transplantation, the appearance of pathology several years in a neurodegenerative environment argues that the initiation factor of pathology had been conferred and affected healthy neurons [45, 46].

In line with this histopathological evidence, more recent work in cell culture and animals models has provided direct indication that α-Syn pathology is transmissible to recipient cells. In particular, it is now clear that brain homogenates enriched in α-Syn pathology can induce Lewy pathology in the CNS of recipient animals. Importantly, the source of this pathology, whether from transgenic mice exhibiting Lewy-like pathology or from MSA or PD patient-derived extracts are capable of initiating pathological formation [47–50]. Indeed, α-Syn itself can propagate pathology as recombinant α-Syn fibrils (or pre-formed fibrils; PFFs) and induce Lewy-like pathology in cultured cells and neurons, as well as in vivo following intracerebral introduction [51–56]. Not only does α-Syn pathology form in these models, but aggregation develops at sites at considerable distances from the injection site in vivo indicating propagation of the misfolding process [53–55].

Interestingly, intramuscular PFF injection also results in CNS pathology in transgenic mice overexpressing α-Syn, suggesting that pathology can transmit via the PNS, although the mechanism responsible remains unclear [57]. Analogous to this, the preponderance of Lewy pathology in deep brain stem nuclei, especially the dorsal motor nucleus of the vagus, seen in early PD is consistent with a spreading process originating in peripheral sites [39]. One location commonly found to harbor α-Syn aggregates in PD patients are gastrointestinal tract neurons, and a recent report suggests that long term risk of PD is significantly reduced following full vagotomy, raising the interesting possibility of this system as potential route of spread [58, 59].

The potential mechanisms by which misfolded α-Syn can initiate and propagate intracellular inclusion formation that is reminiscent of human synucleinopathies have been reviewed in detail elsewhere [5, 60–62] and appears to be predicated on three major components: the generation of misfolded α-Syn species, internalization into a permissive cellular environment/compartment, and engagement with the endogenously expressed α-Syn pool.

2.1 Internalization of extracellular α-Syn

Among neurons, endocytosis appears to be a chief mechanism mediating misfolded α-Syn entry into neurons, although the precise steps and whether other internalization pathways play a role is an active area of research [52, 63]. Blocking endocytosis with a dynamin dominant negative mutant prevents cell to cell transfer of pathology [64]. It is not clear if this is mediated by a selective uptake mechanism, e.g. a specific receptor that binds α-Syn, although α-Syn aggregates appear to have an affinity for heparan sulfate proteoglycans on neuronal cell surface like tau fibrils [65]. In line with these properties, α-Syn internalization has also been described in multiple cell types, and evidence suggests that microglia, astrocytes and oligodendrocytes are capable to internalizing α-Syn [66–70]. Several commonly used cell lines have also been shown to internalize α-Syn [51, 71–73]. This provides evidence as to how glia cytoplasmic inclusions (GCIs) could form in MSA, but it is unusual that these inclusions would be able to form because neither cell type expresses much α-Syn [66, 68]. Another potential mechanism that could spread pathology are nanotunnels between cells which have been shown to be capable of spreading prion protein, but this has yet to be reported for α-Syn [74].

The manner in which α-Syn is released from cells remains unknown as well. α-Syn is found in vesicles released by exocytosis and in several body fluids, such as CSF and plasma [75, 76]. α-Syn secretion continues when ER-Golgi transport is blocked, ruling out conventional exocytosis as the primary mechanism of α-Syn release [75, 77]. Other non-conventional forms of exocytosis are being explored as the mediator of α-Syn spread including exosome release and exophagy, an autophagosome mediated exocytosis [78–81].

2.2 Multiple neuronal populations are permissive to pathological α-Syn propagation

Mice, rats as well as non-human primates have been found to develop pathology and nigrostriatal neurodegeneration after injection of PD patient brain lysate into the striatum further illustrating that pathology and DA neuron toxicity are transmissible [48, 56]. Targeting PFFs to separate CNS nuclei in WT mice also gives rise to distinct spreading patterns for each injection site [82]. Thus, it is clear that multiple cell populations in the CNS are permissive to seeding with exogenous misfolded α-Syn species. This data also indicates that pathology spreads in an anatomically dependent manner. Furthermore,in vivo data suggests that pathology spreads in a retrograde manner to synaptically connected brain regions, and this is unlikely to be mediated by cell death [53, 83, 84]. Pathology has also been observed to be trafficked in vitro both towards and away from the soma [85, 86]. The terminology of retrograde vs. anterograde should be used cautiously as pathology propagation mechanisms are ill-defined and may not conform to traditionally defined modes of axonal transport. Moreover, it is possible that different species of α-Syn (e.g., internalized misfolded vs. converted endogenous species) may undergo different modes of trafficking/transport (i.e., propagation may be a separate but simultaneous event), although pathology is capable of propagating both towards and away from the soma in cultured neurons [61].

The range of conformational species capable of inducing pathology remains a topic of debate. Indeed, aggregates generated under different conditions or isolated from different synucleinopathies have been reported to induce distinct phenotypes, leading to the notion that multiple strains exist. When α-Syn is fibrilized in vitro under stringent conditions, distinct α-Syn fibrils with different biochemical and biophysical properties can be generated. These different fibril strains are able to cause different phenotypes in vivo as well [67, 87]. In addition, α-Syn fibrils formed after serial passaging have been found to gain novel properties and co-aggregate other aggregation prone proteins such as tau, which is implicated in Alzheimer’s and other disorders [88]. This raises the possibility that PD and other synucleinopathies may be caused by different strains of fibrillized α-Syn in humans, although this is not yet definitive.

3. Toxicity mediated by α-Syn misfolding

It is unclear if LB formation precedes cell loss, but the appearance of α-syn aggregates within a neuronal population is highly correlated with the onset of neuronal loss [38, 89, 90]. In PD patients, cell loss in the SNis also highly correlated to worsening of symptoms, and disease progression is associated with changes in network activity in several brain regions including the subthalamic nucleus and globus pallidus [91, 92]. However, the degree to which these two variables correlate and their causality is still contentious.

The sequence of events is somewhat clearer in experimental models and neurons treated with α-Syn PFFs is that PFF-injected mice exhibit pathology formation before the onset of cell loss suggesting that pathological species lead to neurodegeneration [52, 53, 93]. Nonetheless, there is considerable debate as to whether inclusions such as LBs act as a protective sink for toxic α-Syn intermediates or that they lead to sequestration of the physiological α-Syn pool, resulting in the loss of normal α-Syn function. Thus, an important question is whether neuroprotection or toxicity (or neither) is enhanced in cells that contain the pathological aggregates. To address this issue, Unni and colleagues used multi-photon imaging to monitor α-Syn pathology development and long term neuronal viability after intracerebral α-Syn PFF injection in mice expressing an α-Syn-GFP fusion protein [94]. As predicted, these animals developed inclusions of α-Syn-GFP that matured and condensed over time. By following the α-Syn-GFP aggregates within individual neurons over time, the authors found that the aggregate-bearing neurons were selectively lost. Their results also showed that while affected neurons were lost over a range of time after inclusion formation, neighboring neurons were consistently preserved, suggesting the lack of a widespread toxic process involving other cells.

Collectively, these data provide several interesting clues into the toxicity of α-Syn aggregation and the cause of neurodegeneration. First, they suggest that α-Syn inclusion formation is necessary for toxicity and that cell death correlates with inclusion maturation. Although it is clear that neurons containing pathology are the ones that are vulnerable, this raises the question of whether fibrillar forms of α-Syn found in pathological aggregates is necessary for neurodegeneration in the susceptible population. Moreover, synaptic and trafficking impairments are detectable soon after pathological seeding when inclusions are confined to axons [95]. Lastly, they suggest that cell death following inclusion formation is likely a primarily cell-autonomous process. α-Syn inclusion induced cell death is also apparent in primary neuronal culture, where microglial responses are limited [52]. Although, this does not preclude the possibility of microglia activating after the onset of neurodegeneration further enhancing cell death.

3.1 A protein of many (ascribed) functions: implications of loss of function

α-Syn is the most studied member of a three member protein family comprised of α, β, and γ synuclein [7, 96]. α-Syn is an 140-amino acid protein comprised of three distinct domains. An amphipathic N-terminal portion containing seven 11-amino acid repeats in thought to interact with membranes by adopting an alpha helical structure when bound [97]. The function of the acidic and proline-rich C-terminal region is unknown, but the presence of several putative phosphorylation and post translational sites points to involvement in regulating α-Syn activity and/or localization [54, 98]. Biophysical studies also suggest that the C-terminus also interacts with the hydrophobic region that forms the core of the α-Syn molecule, preventing its self-assembly into the beta-sheet rich amyloid fibrils in LBs and GCIs [99, 100].

Although it is found in a number of tissues, α-Syn is most highly expressed in both peripheral and central neurons [101]. At the intracellular level, the highest levels of α-Syn are colocalized to pre-synaptic terminals, consistent with its purported involvement in regulating synaptic vesicle cycling and endocytosis [101–104]. α-Syn has been shown to facilitate and stabilize SNARE complex formation, and overexpression of WT α-syn in cultured neurons leads to decreased exocytosis in both hippocampal and DA neurons [105, 106]. Interestingly, apart from modulation in DA release and recycling pool homeostasis, mice lacking α-Syn show no noticeable phenotype, possibly owing to redundancy of the other synucleins or other compensatory mechanisms [107–110]. Indeed, mice lacking α-, β-, and γ-syn show age-related neurodegeneration and endocytosis abnormalities [111, 112]. Acute knockdown of α-Syn by means of adeno-associated virus expressed RNAi also promotes degeneration in SN neurons [113].

In addition, α-Syn has been reported to be localized to the cytosol, nucleus, the mitochondria associated membrane of the ER, and the inner membrane of mitochondria, suggesting that it may be actively trafficked between different compartments [114, 115]. Additional studies have found that α-syn can affect nuclear protein function [116–118]. α-Syn’s molecular weight is below the nuclear pore cut off allowing α-syn to diffuse from the general cytosolic population into the nucleus without a prototypical NLS. Other putative functions for α-syn involve mitochondrial maintenance, consistent with reports that α-Syn binds to mitochondria, and perturbation of α-syn levels can impair mitochondrial function [119–122]. Nonetheless, many of these functions ascribed to α-Syn remain too poorly defined, and whether their loss contributes to toxicity remains to be elucidated.

3.2 Misfolded α-Syn as toxic species

In addition to the prototypical 12–15 nm wide amyloid fibrils found in LBs, multiple molecular species of various sizes have been reported, including short fibril-like intermediates (protofibrils) and spherical or annular oligomers ranging from ~25 to several hundred α-Syn molecules [123, 124]. Toxic oligomers contain a β-pleated sheet structure and are much smaller compared to aggregates and are variable in size. Oligomers are believed to be on the pathway between α-Syn monomer and fibrils, but off-pathway oligomers have been described to cause toxicity [125, 126]. It is also possible that as α-Syn becomes fibrillar and forms inclusions, the concentration of intermediates (i.e. oligomers) may increase which could increase toxicity [127]. Toxicity or mechanism thereof has been controversial, although a common theme is the disruption of membranes. Winner et al. showed that α-Syn mutants that preferentially formed oligomers resulted in enhanced DA neuron loss when overexpressed in rat midbrain, and α-syn oligomers are capable of forming pores in membranes and cause cell toxicity and death due to increased calcium permeability [128–131].

The topic of oligomers as the toxic species is still contentious because membrane permeabilization may not be specific to oligomers and may only be transient, which would suggest that what is actually being observed is α-syn transiently binding to membranes [132, 133]. Moreover, the unstable/transient nature of oligomeric α-Syn has also precluded their isolation and in-depth characterization. The recent work into oligomer and fibril toxicity suggests a gain of toxic function mechanism, but this does not exclude the possibility of loss of α-syn function further increasing toxicity. These two options need not be mutually exclusive, and this remains an active area of debate.

The α-Syn aggregation and cell loss seen after seeding pathology with α-syn PFFs is highly dependent upon the production of α-Syn within neurons. Importantly, neither cell loss nor the induction of pathology occurs in α-Syn^−/− mice or primary neurons [52, 53]. We have also observed that as primary neurons mature they develop pathology at a much faster rate compared to younger neurons, which is most likely due to primary neurons producing more α-syn as they age in culture [52]. Interestingly, it has been noted that the GABAergic neurons are resistant to α-Syn aggregation in primary hippocampal cultures after PFF addition, due to their lower levels of α-Syn expression compared to glutamatergic neurons despite comparable capacity to internalize α-Syn [134]. Similarly, M83 hemizygous mice expressing the A53T α-Syn mutant injected intracerebrally with PFFs show delayed onset of symptoms compared to injected homozygous mice [47, 49]. It is worth noting that symptom-free survival in M83 hemizygous mice was nearly double that of the homozygous counterparts, but their rate of decline was similar suggesting that the increased α-syn expression in the M83 homozygous mice increased the rate of nucleation and not neurodegeneration. Supporting this is the observation that patients with α-Syn triplications develop disease at an earlier age compared to patients with α-Syn duplications [23].

4. Origins of misfolded α-Syn

Despite the expanding literature on cell-to-cell transfer of α-Syn, much less is known about how misfolded α-Syn species initially arise. The ubiquitous and robust expression of α-Syn in neurons makes them the most probable location for this to occur. Impairments in protein degradation and quality control machinery directly increase intracellular α-Syn concentration, augmenting the rate of both cell autonomous α-Syn aggregate formation and the risk of seeding by exogenous α-Syn.

However, the localization of α-Syn may be as important as its quantity. Most groups report that α-Syn is mostly a natively unfolded monomer within the cell using both denaturing and non-denaturing conditions and that α-Syn is only weakly associated with synaptosomal and mitochondrial fractions [112, 135–137]. This contrasts with reports that α-Syn exists as a membrane bound multimer and that stable tetramers can be isolated under non-denaturing conditions [138–140]. Given the affinity of α-Syn for phospholipids and high curvature membranes, this discrepancy could reflect interactions between α-Syn and membranes that are highly transient. Indeed, cross-linking appears to stabilize a multimeric membrane bound pool and SNCA mutations known to cause familial PD cluster within a small stretch of the N-terminal region, suggesting that interactions with membranes is vital [138–143]. These mutations, as well as directed mutations in the imperfect repeats preceding the core hydrophobic domain, reduce membrane binding through disruption of α-Syn multimers [144, 145].

Increased α-Syn dissociation is thought to result in the accumulation of unfolded species in the cytoplasmic compartment shifting the α-Syn equilibrium towards the formation of oligomeric and/or fibrillar species. Although several familial PD α-Syn mutants show accelerated in vitro aggregation kinetics (e.g., E46K, H50Q, A53T), fibrillization in others are reduced (A30P, G51D), indicating that both increased levels of unstructured α-Syn and aggregation propensity factor in the generation of misfolded fibrillar species [146–149]. In line with this, artificial α-Syn mutants with compromised membrane binding also preferably segregate into the soluble unbound pool. Importantly, overexpression of these mutants in vivo greatly enhances toxicity in SN DA neurons, although it remains to be seen whether wildtype α-Syn also undergoes dissociation into a more aggregation-prone state at levels that support misfolding [150].

Although modest accumulation is observed in the brains of transgenic animals overexpressing α-Syn, the dramatic increase in pathological α-Syn following introduction of either PD brain extracts or recombinant α-Syn fibrils suggests that normal α-Syn located within membranes are vulnerable to conversion by pathologic species. This accelerated conversion is also apparent in models of α-Syn seeding using non-transgenic rodents as well as non-human primates indicating that fibrillar, and possibly oligomeric intermediates, are capable of destabilizing native α-Syn [48, 52, 54, 56].

5. Pathophysiology of α-Syn misfolding

Similar to the role α-Syn oligomers play in disease, considerable debate exists regarding the cellular stressors arising from α-Syn misfolding that contribute to neurodegeneration. Nonetheless, work from both genetic and fibril-injection based models of synucleinopathies have provided fresh insights to the knowledge derived from more traditional, mainly neurotoxin (e.g., MPTP, rotenone, and 6-OHDA) dependent models. A particularly interesting observation is that α-Syn appears to modulate the toxicity in these latter models. For example, mice lacking α-Syn expression show relative resistance to MPTP induced degeneration of midbrain DA neurons, further highlighting a role for α-Syn in degeneration and that these approaches may intersect at the stressor pathways they activate [151]. Several different cell stressors have been implicated by both neurotoxin based PD models and genetic models to cause neurodegeneration. Among these are ER stress and the unfolded protein response (UPR), oxidative stress and mitochondrial dysregulation, calcium homeostasis dysregulation, and neuroinflammation.

5.1 Endoplasmic reticulum stress

ER stress and activation of the unfolded protein response have been observed in various cell culture and mouse models of PD where α-Syn aggregation is present, including transgenic mice expressing human α-Syn with the A53T mutation [152, 153]. In both the MPTP and 6-OHDA models of PD upregulation of markers such as CHOP and ATF4 also point to ER stress. Genetic deletion of CHOP can prevent neurodegeneration in the 6-OHDA model, although this same effect did not occur in the MPTP model [154]. In addition, treatment with salubrinal, an inhibitor of ER stress, can alleviate motor symptoms and cause neuroprotection [152]. A downstream consequence of ATF4 and CHOP activation is the increase of Trib3, and knockdown of this pro-apoptotic product reduces ER stress and attenuates neuron loss in both neurotoxin and PFF based models [155].

In PD post mortem patient samples the presence of several key markers, including phosphorylated PERK are also consistent with ER stress and UPR activation in the face of increased misfolded α-Syn [156]. Several genetic mutations that cause familial PD are known to affect protein degradation and trafficking. Chief among these are the GBA, VPS35, ATP13A2, and LRRK2 mutations which affect either lysosomal function or ER trafficking, and there is evidence that VPS35 may interact with α-Syn [34, 157–159]. ER-Golgi trafficking dysfunction has also been noted in the PFF model in multiple cell types [51, 95] while overexpression of Rab1, a regulator of ER-Golgi trafficking in yeast, or its homologs leads to rescue from cell death in PD models [160]. Induced pluripotent stem cells derived from PD patients with genetic mutations also develop ER stress which is alleviated by the ubiquitin ligase Nedd4, further implicating proteosomal/ER stress in PD, and enhanced Nedd4 activity in attenuating this dysfunction [161, 162]. It is unknown if ER stress and UPR activation are a cause or effect in PD pathogenesis, although the evidence presented suggests both.

5.2. Oxidative Stress

As with ER stress, it is unclear if oxidative stress represents a cause and/or effect in PD pathogenesis. Compared to controls, brain tissue from PD patients contain altered levels of antioxidant proteins, such as GPX4 and glutathione, suggesting that this mechanism may play a role in disease [163]. In addition, mutations in Parkin and PINK1, which maintain mitochondrial health, cause autosomal recessive forms of PD [164–166]. Even though knockout of these genes in mice do not result in α-Syn aggregation, increased mitochondrial stress has been observed in these mice indicating that they play a role in maintaining neuronal health [167, 168]. Interestingly, α-Syn null mice are also protected from DA neuron loss following treatment with the mitochondrial complex I inhibitor MPTP suggesting that α-Syn mediates toxicity [169]. Consistent with this, cultured DA neurons exposed to α-Syn fibrils show elevations in both mitochondrial and cytoplasmic oxidative stress [170]. More recently, α-Syn has been shown to be localized with mitochondrial associated ER, suggesting α-Syn may have a function in mitochondrial homeostasis and disruption of this could contribute to ER stress and mitochondrial dysfunction and providing a link to PD pathogenesis and neurodegeneration of DA neurons [114].

5.3 Calcium buffering and vulnerability within DA subpopulations

Calcium ions (Ca⁺⁺) are used extensively for cell signaling but imbalances can also be a potential source of toxicity [171]. Furthermore, it is known that multiple neuronal populations affected in PD show a combination of low Ca⁺⁺ buffering capacity and broad action potentials, leaving them vulnerable to increased cytoplasmic Ca⁺⁺ levels [172, 173]. In particular, SN neurons utilize Cav1.3 Ca⁺⁺ channels for their rhythmic pacemaking activity, exposing them to higher calcium conductance relative to neurons in the nearby ventral tegmental area (VTA) [174]. Blockers that prevent Cav1.3 mediated Ca⁺⁺ entry protect from MPTP but this is not clear for α-Syn PFF models [175, 176]. Curiously, Ca⁺⁺ toxicity in DA neurons also appears to be α-Syn-dependent [177].

Because the ER represents a significant reservoir for intracellular Ca⁺⁺, it is possible that ER stress could be a source of Ca⁺⁺ toxicity in PD. For example disruptions in calcium homeostasis has been shown in midbrain neurons derived from induced pluripotent stem cells from patients bearing GBA mutations [178]. Higher Ca⁺⁺ conductance has been linked to increased oxidative stress [179]. It is possible that a vicious cycle involving a challenge of ER Ca⁺⁺ storage capacity and the reliance on voltage gated Cav1.3 channels leads to elevated Ca⁺⁺ levels that trigger the demise of SN, and possibly other, neurons.

5.4 Neuroinflammation

It has been suggested that neuroinflammation from activated microglia may increase neuronal toxicity. It is known that microglia are activated in areas of neurodegeneration in both post mortem PD tissue and in vivo mouse models of PD [49, 55, 180, 181]. PD patients develop increased pro-inflammatory cytokines in their CSF, such as TNF-α and IL-1β [182, 183]. Oligomeric α-Syn has been shown to bind TLR2, β1-integrin, and CD11b, which can activate microglia [68–70]. It is currently debated whether microglia in PD are actively inducing neurodegeneration, have a more benign role of cleaning the cellular debris left from dying cells, or possibly both. It has been shown that those neurons that develop pathology eventually are lost where the surrounding neurons are spared in PFF models suggesting that cell death is cell autonomous due to the pathological aggregate and not a reactive extracellular mileu [94].

6. Neuron-specific vulnerability

If α-Syn misfolding represents a driving pathogenic event, as suggested by clinical and experimental evidence, then the selective pattern of degeneration that characterizes PD would suggest that certain cell subpopulations possess an enhanced susceptibility to acquire or develop α-Syn pathology along with elevated vulnerability to the induced toxicity (Figure 1). SN DA neurons provide a prototypic example of how these factors might converge in a susceptible population. Firstly, DA neurons carry enormous metabolic demands due to their high-frequency autonomous firing resulting in greater production of oxidative stress and increased calcium entry into the cell for the release of synaptic vesicles [171, 179, 184]. Unlike VTA neurons and other populations that express calcium binding proteins (e.g. calbindin), SN neurons, particularly in the ventral tier, show a paucity of these buffers [174]. This, together with their susceptibility to elevated calcium levels due partly to a dependence on Cav1.3 channels for their pacemaking activity furthers their exquisite sensitivity to perturbations in oxidative respiration (e.g., complex 1 inhibition). In line with the view that DA neurons are especially sensitive to mitochondrial impairment, mutations in mitochondrial DNA or PINK1 and Parkin (two crucial components in mitophagy) are also associated with PD. The bioenergetic demands of DA neurons are further pushed by the need to maintain elaborate and high density of connections with target neurons. It is likely that perturbations in the energy balance disrupt DA neuron arbors leading to the loss of synapses, and in turn make it susceptible to loss of trophic support that likely helps sustain the neurons [185, 186].

Figure 1. Spread and effect of α-Syn pathology.

Proposed sequence of pathological α-Syn transmission. Pathology is initially transmitted between neurons and propagates primarily in a retrograde manner. Resistant populations of neurons (top row) are either capable of degrading the seeds without forming pathology or have increased capacity to survive and function with pathological inclusions. Vulnerable populations (middle row) exhibit α-Syn aggregates that grow and mature over time. These aggregates are capable of disrupting normal neuronal function (yellow-green neurons) by oxidative stress, ER stress, or other potential stress mechanisms. Diseased neurons containing inclusions may be capable of further pathological seeding. After a certain level of cell stress is reached, the diseased neurons undergo cell death (yellow-green neuron with dashed outline).

Given the recent body of evidence suggesting pathological α-Syn transmission, these neurons are at high risk of being exposed to seeding species due to their large number of synapses and poorly myelinated axons. The high levels of α-Syn enriched in processes (especially synaptic terminals) further increases this risk. Consistent with this, over 90% of PD α-Syn pathology is present as Lewy neurites, rather than LBs in the soma [187]. Axons also represent the initial sites for the accumulation of hyperphosphorylated α-Syn inclusions in cultured hippocampal neurons suggesting that synaptic connections represent a general “hot spot” for pathology in other types of neurons [52].

It has also been argued that elevated Ca⁺⁺ levels may also increase the rate of endocytic and secretory events, major routes hypothesized for pathologic α-Syn entry and exit, respectively, and making neurons prone to seeding aggregation. Possibly exacerbating this is the observation that DA neurons exhibit robust α-Syn expression which increases with age in the SN of both humans and non-human primates, a phenomenon that is not observed in other DA neuronal populations that are relatively spared in PD, such as the VTA [188].

Intriguingly, many of these above properties also apply to a long list of neuron subpopulations selectively affected in PD including those in the locus coeruleus, raphe nucleus, dorsal motor nucleus of the vagus, pedunculopontine nucleus, nucleus basalis of Meynert, and enteric neurons in the large intestine. Another commonality between several of them is their monoaminergic neurotransmitter phenotype. In particular, catecholamines are highly reactive and would predispose cells that contain high levels of them to dysfunction or death due to oxidative stress [184, 189]. Cells that contain catecholamine neurotransmitters also can contain neuromelanin (NM) which is believed to be a protective mechanism preventing excess oxidative stress due to catecholamine production [190]. NM is a sign of oxidative stress and can sequester metal ions like iron which can contribute to the Fenton reaction and lipid peroxidation [191]. Supporting this is an age associated increase in nigral NM content, and humans, the only species known to develop PD, tend to develop much larger stores of NM compared to non-human primates [192]. More recently, it has been shown that catecholaminergic neurons are predisposed to MHC I induction compared to other neuronal populations, and that cell death could occur in the presence of the appropriate Cytotoxic T cells making this population prone to neuronal loss [193].

7. Conclusion

The past several years have witnessed a dramatic expansion in our knowledge regarding the role of α-Syn in the pathogenesis of PD and related proteinopathies. Recent work clearly suggests that rather than a single dominant factor, the causes of α-Syn pathology is multifaceted. Furthermore, these processes interact with inherent vulnerabilities of certain neurons to give rise to the observed degeneration and symptoms. These factors vary across neuron populations in the central and peripheral nervous systems, and likely underlie some of the variability between the synucleinopathies. Pathological α-Syn behaves as a prion-like agent, capable of templating the conversion of intracellular α-Syn and providing a route for the propagation of the pathological process into unaffected neurons. The rate of pathology induction is partially dependent on the total amount of α-Syn, which can vary across neuronal populations. Following this is the onset of neuronal dysfunction and then neurodegeneration of those neurons that contain sufficiently large pathological aggregates (Figure 1). Understanding these areas and how they interact will be key to developing disease modifying therapeutics to treat these patients.