α-Synuclein: Multiple pathogenic roles in trafficking and proteostasis pathways in Parkinson’s disease

Abstract

Parkinson’s disease (PD) is a common age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the midbrain. A hallmark of both familial and sporadic PD is the presence of Lewy body inclusions composed mainly of aggregated α-synuclein (α-syn), a presynaptic protein encoded by the SNCA gene. The mechanisms driving the relationship between α-syn accumulation and neurodegeneration are not completely understood, although recent evidence indicates that multiple branches of the proteostasis pathway are simultaneously perturbed when α-syn aberrantly accumulates within neurons. Studies from patient-derived midbrain cultures that develop α-syn pathology through the endogenous expression of PD-causing mutations show that proteostasis disruption occurs at the level of synthesis/folding in the endoplasmic reticulum (ER), downstream ER-Golgi trafficking, and autophagic-lysosomal clearance. Here, we review the fundamentals of protein transport, highlighting the specific steps where α-syn accumulation may intervene and the downstream effects on proteostasis. Current therapeutic efforts are focused on targeting single pathways or proteins, but the multifaceted pathogenic role of α-syn throughout the proteostasis pathway suggests that manipulating several targets simultaneously will provide more effective disease-modifying therapies for PD and other synucleinopathies.

Parkinson’s disease: clinical and pathophysiologic hallmarks

Parkinson’s disease (PD) is a common age-related neurodegenerative disorder, affecting 1% of the population over 60 and greater than 4% over 85 (Hirsch and others 2016; reviewed by Tanner and Goldman 1996). Clinical features of PD include bradykinesia, tremors, postural instability, and rigid movements (Gelb and others 1999). There are both familial and sporadic instances of this disease, making up about 10% and 90% of cases, respectively (reviewed by Lin and Farrer 2014). Importantly, in both etiologies, symptoms are attributed to the degeneration of midbrain dopaminergic neurons (Braak and others 2003; Fearnley and Lees 1991; Iacono and others 2015), and the major pathologic hallmark is the presence of neuronal Lewy body (LB) inclusions (Spillantini and others 1997). LBs are insoluble inclusions composed mainly of filamentous alpha-synuclein (α-syn) but also contain neurofilaments, ubiquitin, lipids, and organelles such as mitochondria and lysosomes (reviewed by Lashuel 2020; Shahmoradian and others 2019). LB pathology is thought to progress in stages, where it is first observed in brainstem and olfactory structures, later progressing to the substantia nigra and finally the neocortex (Braak and others 2003). In addition to motor symptoms, most PD patients experience progressive cognitive impairments; the probability of developing nonmotor symptoms, including dementia, has been proposed to correlate with increased spreading of LB pathology into cortical regions (Braak and others 2005). Clinical and pathologic heterogeneity in PD has been well documented. For example, age at onset and degree of cognitive impairment between PD patients vary greatly. Most patients (~80%) will eventually develop dementia with a mean onset of 10 years after PD diagnosis (known as PD dementia [PDD]). However, the time of onset is highly variable, with some patients developing dementia shortly after PD diagnosis and others remaining cognitively stable for 20 years (reviewed by Aarsland and Kurz 2010). If dementia is present before, concomitantly, or within a year of PD diagnosis, it is classified as dementia with Lewy bodies (DLB) (reviewed by McKeith and others 2017). It has been proposed that PD, PDD, and DLB are the same disease but with different rates of progression along a spectrum, but the factors that modulate this progression are unknown. Genetic studies have suggested that variants in lysosomal GBA1 are involved in the rate of progression of cognitive deficits in PD patients, since carriers are more likely to develop early-onset dementia (Alcalay and others 2012; Brockmann and others 2011). Beyond the synucleinopathies mentioned above, Lewy body pathology is observed in other neurodegenerative diseases, including multiple system atrophy (MSA), and neuronopathic subtypes of the lysosomal storage disorder Gaucher’s disease (GD) (reviewed by Koga 2021; Wong and others 2004).

α-Synuclein: physiologic role and pathologic contribution to PD

α-Syn is highly expressed in neurons, most notably in the striatum, hippocampus, thalamus, cerebellum, and neocortex (reviewed by Burré and others 2018). Despite the significant role it plays in PD pathogenesis, the physiologic functions of this presynaptic protein are not fully understood. However, given its localization to presynaptic terminals (Maroteaux and others 1988) and its interaction with synaptic proteins (Burré and others 2014), evidence from both in vivo and in vitro studies suggests α-syn contributes to various presynaptic functions, including neurotransmitter release (Abeliovich and others 2000; Gureviciene and others 2007; Nemani and others 2010; Yavich and others 2005, 2006) and vesicle pool maintenance (Murphy and others 2000). Although little is known about the physiologic role of α-syn in intracellular protein trafficking, previous work has shown that the native protein can participate in membrane remodeling (Kamp and others 2010; Nakamura and others 2011) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly (Burré and others 2010; Chandra and others 2005). Interestingly, one study overexpressing wild-type (WT) α-syn in PC12 cells reported an enrichment in docked secretory vesicles, suggesting an α-syn–induced impairment in vesicle-membrane fusion (Larsen and others 2006). While there is ongoing work dedicated to understanding the physiologic role of α-syn in the brain, for the purposes of this review, we will focus on mechanisms of α-syn toxicity in PD. An in-depth review on recent findings pertaining to the primary function of α-syn can be found elsewhere (reviewed by Sulzer and Edwards 2019).

Importantly, in addition to the growing body of work delineating the mechanism of α-syn toxicity on essential cellular processes, strong genetic evidence exists directly linking both mutant and WT SNCA to PD pathogenesis. In the past 25 years, several familial-linked point mutations have been identified. One of the most well-studied is the SNCA A53T mutation (Polymeropoulos and others 1997). The A53T mutation, as well as most other autosomal dominant pathogenic SNCA variants associated with PD (e.g., A30P [Kruger and others 1998], E46K [Zarranz and others 2004], and H50Q [Proukakis and others 2013]) can increase the propensity to aggregate into oligomers or fibrils (Conway and others 1998, 2000; Cullen and others 2011; Ghosh and others 2013; Greenbaum and others 2005; Rutherford and others 2014). This, in addition to the autosomal dominant inheritance pattern found within these families, suggests these variants likely elicit a gain-of-toxic function effect on α-syn.

In addition to point mutations, an accumulation of WT α-syn is also sufficient to trigger the disease cascade. While rare, some familial PD cases are caused by duplication or triplication of the genomic region that harbors WT SNCA (Chartier-Harlin and others 2004; Singleton and others 2003). α-Syn triplication (SNCA-3X) patients carry four copies of the SNCA genomic region, accumulate insoluble α-syn, and exhibit severe parkinsonism and dementia at 20 to 30 years of age. Patients who carry the duplication mutation have three total copies and experience milder initial symptom onset, which occurs 30 to 40 years later in life and more closely resembles sporadic PD (reviewed by Singleton and Gwinn-Hardy 2004). This demonstrates the dose-dependent nature of α-syn toxicity in PD pathogenesis. Multiple transgenic animal models have shown that neuron-restricted α-syn overexpression is sufficient to induce synuclein pathology and neurodegeneration (reviewed by Dawson and others 2010), and the addition of preformed fibrils of α-syn can corrupt the conformation of physiologic conformers, inducing aggregation and toxicity (Luk and others 2012; Volpicelli-Daley and others 2011). Studies have documented that endogenous, physiologic α-syn can exist in multiple conformations, including monomers, tetramers, or higher-order oligomers (Bartels and others 2011; Burré and others 2014; Fauvet and others 2012; Weinreb and others 1996; Zunke and others 2018). Upon binding to acidic phospholipids, the N-terminus of monomeric α-syn assumes an alpha-helical structure (Davidson and others 1998). The N-terminus is also responsible for interacting with components of processing (P)-bodies that maintain mRNA stability under physiologic conditions (Hallacli and others 2022). Recent work shows that a buildup of α-syn monomers could exhibit toxicity, through excessive membrane interactions or P-body components (Dettmer and others 2017; Hallacli and others 2022). Taken together, these data suggest that while multiple conformations of α-syn could be toxic, excessive amounts of α-syn are detrimental to neurons. Therefore, methods that prevent its accumulation hold promise as a therapy for PD. While the exact pathogenic mechanisms of α-syn are unknown, the past few decades have revealed perturbations in protein trafficking, and intersecting lysosomal degradation pathways contribute to neurodegeneration and may converge through the multiple toxic functions of α-syn.

Intracellular protein trafficking and synuclein toxicity in PD

Overview

Strong evidence implicating impaired intracellular trafficking in PD pathogenesis comes from PD genetics, which have identified several distinct risk loci in multiple trafficking components and pathways that converge on lysosomal clearance, including macroautophagy, endocytosis, and mitophagy (reviewed by Abeliovich and Gitler 2016). In particular, the endocytic/retromer system appears to harbor many PD-associated variants in genes that are involved in multiple trafficking stages, such as cyclin G–associated kinase (GAK) and DNAJC6/auxilin that function in clathrin uncoating (Dumitriu and others 2011; reviewed by Eisenberg and Greene 2007; Olgiati and others 2016; Ungewickell and others 1995). RAB29/RAB7L1 on the PARK16 locus normally mediates retromer function and Golgi-lysosome trafficking, and its loss causes cargo mistrafficking, lysosomal function, and neurodegeneration (MacLeod and others 2013). RAB29 functionally interacts with two other important genes discovered to cause monogenic PD, vacuolar protein sorting ortholog 35 (VPS35) (Vilarino-Guell and others 2011; Zimprich and others 2011) and leucine-rich repeat kinase 2 (LRRK2) (Zimprich and others 2004). Although the mechanisms are not completely understood, VPS35 mutations can induce lysosomal dysfunction by perturbing endosome-Golgi trafficking of the mannose-6-phosphate receptor (M6PR), which is required for delivering most hydrolases into the lysosomal compartment (Tang and others 2015; reviewed by Williams and others 2022). Loss of RAB29 induces retromer breakdown and depletion of VPS35, indicating their functional interaction in the retromer pathway (MacLeod and others 2013). LRRK2 is a kinase with multiple proposed roles in endomembrane trafficking and can disrupt vesicular transport through phosphorylating Rab GTPases, which are critical regulators of membrane organization and protein sorting (reviewed by Cookson 2016; Steger and others 2016). LRRK2 can phosphorylate RAB29 and functionally interact in the Golgi-lysosomal and retromer pathways (Liu and others 2018; MacLeod and others 2013). Several studies have indicated that LRRK2 can directly or indirectly influence the autophagic-lysosomal pathway (ALP) (Alegre-Abarrategui and others 2009; Gomez-Suaga and others 2012; reviewed by Madureira and others 2020; Manzoni and others 2013; Plowey and others 2008; Schapansky and others 2018), including several that specifically examine the relationship between LRRK2 and chaperone-mediated autophagy (CMA) (di Domenico and others 2019; Ho and others 2020; Orenstein and others 2013). Together, these findings are indicative of the mechanistic convergence with other disease-causing PD mutations, variants, and toxic mechanisms of α-syn (reviewed by Klein and Mazzulli 2018).

Beyond the genetic evidence implicating later-stage membrane trafficking, the early secretory pathway—notably, protein folding capacity, endoplasmic reticulum (ER) quality control, and ER-Golgi trafficking—has also been linked to α-syn toxicity and disease progression (reviewed by Abeliovich and Gitler 2016; Cooper and others 2006; Cuddy and others 2019; Schondorf and others 2014; Stojkovska and others 2022). In PD models from yeast to patient-induced pluripotent stem cell (iPSC)–derived neurons, α-syn accumulation has been shown to interfere with trafficking at several steps. The relative contribution to the disease cascade, especially of issues relating to ER dyshomeostasis, remains unclear. As discussed below, perturbations in the early secretory pathway were one of the first cellular phenotypes observed in α-syn transgenic mice or cells overexpressing cytosolic α-syn and have been reproduced by multiple groups (Cooper and others 2006; Gosavi 2002; Masliah and others 2000). Collectively, studies show that disruption of multiple pathways beyond ER-Golgi is important for PD pathogenesis, including mitochondrial and other branches of the proteostasis pathway (reviewed by Nguyen and others 2019). Recent work from our group also shows that α-syn accumulation perturbs multiple proteostasis steps and that rescuing multiple pathways simultaneously leads to a more efficacious rescue strategy in patient cultures (Stojkovska and others 2022). Here, we will discuss the basic steps of intracellular protein trafficking with a focus on the major proteostasis branches and review seminal and recent evidence indicating that α-syn disrupts this process.

Protein targeting and folding in the ER

The ER plays multiple critical proteostatic roles in the early secretory pathway and is responsible for the synthesis, folding, and processing of one-third of proteins within a cell. It is a major intracellular calcium reservoir and responsible for the production of secretory, endomembrane, plasma membrane, and lysosomal proteins. Proteins can be targeted to the ER in three main routes. The first discovered and best described pathway involves cotranslational entry of polypeptides that are synthesized on ribosomes on the rough ER, followed by entry into the ER lumen through the Sec61-containing translocon complex (Görlich and Rapoport 1993). A polypeptide chain tagged for ER processing is correctly transported because its ER signal sequence, often a string of N-terminal hydrophobic residues, emerges from a ribosome and binds to a large complex called the signal-recognition particle (SRP) (Walter and Blobel 1980), which cycles back and forth between the cytosol and ER membrane. The protein is then recognized by an ER-membrane SRP receptor (Meyer and others 1982), a complex of transmembrane proteins located in the rough ER, which will then deliver cargo to an available protein translocator. This relocation allows for entry across the ER membrane through the hydrophilic pore of the translocator (reviewed by Hegde 2022; reviewed by Zimmermann and others 2006). A second way that proteins can be targeted to the ER is post-translationally and SRP independently, through the transmembrane recognition complex of 40 kDa (TRC40). TRC40 is involved in targeting a specific class of proteins containing C-terminal transmembrane do-main, called tail-anchored (TA) proteins. The TA protein is released from the ribosome, followed by interaction with TRC40 in the cytosol, which then targets the protein for insertion into the ER (Stefanovic and Hegde 2007). Finally, a third route to the ER acts as an auxiliary transport system that can compensate for the loss of the SRP or TRC40 pathways. This pathway involves SRP-in-dependent targeting proteins (SNDs) that function as membrane-bound receptors for nascent TA proteins (Aviram and others 2016; Haßdenteufel and others 2017). The SND pathway also requires cytosolic chaperones, including HSP70 and HSP40, that prevent aggregation and assist in the targeting of proteins to the ER membrane (Rabu and others 2008). An in-depth review on these topics, beyond the basics presented here, can be found elsewhere (reviewed by Fewell and Brodsky 2000).

Within the ER, proteins are modified by N-linked glycosylation that aids in their folding, sorting, and exit from the ER (reviewed by Cherepanova and others 2016). This process is governed by several molecular chaperones, the most abundant and well-studied being calnexin (CANX), calreticulin (CALR), GRP78 (HSPA5), GRP94 (HSP90B1), and protein disulfide isomerases (PDIs). Both calnexin and calreticulin are lectin-type chaperones that interact with monoglucosylated forms of N-glycans on partially folded proteins and retain unfolded proteins in the ER until they are properly folded (Peterson and others 1995; Rajagopalan 1994). Calnexin and calreticulin promote folding and prevent premature degradation of partially folded intermediates through their selective interaction with monoglucosylated N-glycans in the ER. Monoglucosylated N-glycans are formed by either trimming of glucose from triglucosylated cores by ER enzymes glucosidase I and II or through the addition of glucose to an already existing mannose core via uridine 5′-diphosphate (UDP)–glucose:glycoprotein glucosyltransferase I or II (UGGT) (Hebert and others 1995; Labriola and others 1995). These enzymes participate in folding cycles by their ability to recognize partially misfolded proteins. While glucosidase trims an oligosaccharide of its terminal sugar group, glucosyltransferase continuously adds it back, but this occurs only to proteins that are not fully folded (Labriola and others 1995). Once a protein is in its native conformation, glucosyltransferase will stop the cycle and the folded protein will be released from the ER chaperone (reviewed by Cherepanova and others 2016). Interestingly, recent studies have indicated that UGGT enzymes display selectivity in their substrates, with UGGT1 preferentially modifying plasma membrane proteins, while UGGT2 modifies lysosomal proteins (Adams and others 2020). Calnexin and calreticulin are the best studied ER lectin-type, calcium-dependent chaperones and are particularly important for the folding of lysosomal beta-glucocerebrosidase (GCase), a critical enzyme involved in PD pathogenesis, as well as other lysosomal hydrolases (Osaki and others 2019; Ou and others 1993; Tan and others 2014). However, additional machinery is involved in the proper folding of N-glycosylated proteins, including ERp57 (PDIA3), that interact with calnexin and calreticulin and catalyzes disulfide bond formation in the ER (Oliver and others 1997). ERdj3 (DNAJB11) interacts with GCase and plays a role in the ER-associated degradation (ERAD) of misfolded wild-type and mutant forms of the protein (Tan and others 2014). Other ER chaperones, including GRP78 and GRP94, are involved in the unfolded protein response and ERAD (see “ER quality control” section). A more comprehensive description of ER chaperones and their role in ER homeostasis and human disease can be found elsewhere (reviewed by Ni and Lee 2007).

There are a number of possible outcomes for a protein following folding: it can be retained in the ER if it is an ER resident protein, continue transport to the Golgi via ER-derived vesicles, or be expelled from the ER and degraded by either the ubiquitin proteasome system (UPS) or the ALP if it is improperly folded (reviewed by Sun and Brodsky 2019).

ER quality control

In the event of protein misfolding and ER stress, tightly regulated quality control mechanisms have evolved to maintain proteostasis known as the unfolded protein response (UPR). The UPR attempts to maintain ER proteostasis by both decreasing protein synthesis while simultaneously up-regulating specific chaperones to aid in refolding ER proteins. In addition, the ER morphologically expands to accommodate the added protein load (reviewed by Walter and Ron 2011). Early studies of ER stress response identified up-regulation of glucose-regulated proteins, including GRP78 and GRP94, that respond to misfolded proteins retained in the ER and aid in their refolding (Kozutsumi and others 1988). GRP78 also regulates the activity of transmembrane stress sensors of the UPR. Under physiologic conditions, GRP78 is constitutively expressed and bound to an auto-inhibiting inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and protein kinase-like ER kinase (PERK) that comprise the three main UPR stress sensor pathways (reviewed by Walter and Ron 2011). In the event of protein misfolding, GRP78 can be released from these sensors when an abundance of misfolded proteins are present to assist in their refolding (Bertolotti and others 2000; Okamura and others 2000; Shen and others 2002). As a result of this disassociation, the UPR is activated and attempts to restore ER homeostasis are initiated by transcriptional activation of chaperones, reduced translation, and ER expansion (reviewed by Ibrahim and others 2019) (Fig. 1). Alternatively, IRE1 can directly detect unfolded proteins and activate the UPR through peptide binding to their luminal domains (Gardner and Walter 2011). Each of the three branches of the UPR pathway uses different mechanisms to respond to stress following their activation. Briefly, PERK causes downstream phosphorylation and inhibition of eukaryotic initiation factor 2 (elF2α), resulting in decreased translation of new proteins (Fig. 1.1) (Harding and others 2001). IRE1 causes X-box-binding protein 1 (XBP1) mRNA spicing, translation of XBP1s, and XBP1 protein translocation to the nucleus where it assists in transcriptionally regulating protein folding genes (Fig. 1.2) (Shen and others 2001; Yoshida and others 2001). Last, ATF6 is trafficked to the Golgi and cleaved (ATF6-N), serving as a transcriptional activator for ER chaperones and other ER machinery necessary for maintaining homeostasis (Fig. 1.3) (Yoshida and others 1998). If protein refolding fails, the cell attempts to eliminate the misfolded protein through a process called ER-associated degradation (ERAD). Here, misfolded proteins in the ER lumen exit the chaperone-mediated folding cycle via their recognition by luminal chaperones including GRP78 (Plemper and others 1997) and ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1). Misfolded proteins are then translocated through a pore embedded in the ER membrane called the derlin pore. Here, improperly folded proteins are ubiquitinated by E3 ligases, effectively tagging them for proteasomal degradation (reviewed by Smith 2011). Importantly, there are both bulk and selective degradative pathways for misfolded proteins and other damaged cargo that function independent of the proteasome (e.g., bulk autophagy and selective ER-phagy). It is well established that autophagy is critical for neuronal health and heavily implicated in PD pathogenesis. We will discuss these pathways in detail below.

Figure 1.

Unfolded protein response (UPR). The three major UPR signaling pathways (1) protein kinase-like ER kinase (PERK), (2) inositol-requiring enzyme 1 (IRE1), and (3) activating transcription factor 6 (ATF6). Each pathway is inhibited by direct binding of GRP78. Under endoplasmic reticulum (ER) stress, GRP78 will dissociate and assist with misfolded proteins inside the ER lumen, resulting in UPR activation. (1) PERK activation results in downstream phosphorylation of eukaryotic initiation factor 2 (elF2α). Inhibition of elF2α decreases the translation of new proteins, thereby assisting in reducing burden to the ER. Further, activating transcription factor 4 (ATF4) mRNA is selectively translated because of PERK activation. ATF4 then translocates to the nucleus, where it assists in the regulation of adaptive stress response genes. (2) IRE1 activation results in X-box-binding protein 1 (XBP1) mRNA splicing and subsequent translation of XBP1s. XBP1 protein translocates to the nucleus and assists in the activation of genes involved in ER-associated degradation (ERAD) and protein folding. (3) Activation of ATF6 allows it to translocate to the Golgi. It is then cleaved (ATF6-N) and serves as a transcriptional activator for ERAD machinery and other chaperones important for maintaining ER homeostasis. Adapted from Walter and Ron 2011. Created with BioRender.com.

If ER quality control mechanisms cannot manage the burden of accumulating misfolded proteins, the adaptive function of the UPR can switch to trigger apoptosis after a prolonged period of failed attempts to correct protein misfolding (reviewed by Colla 2019; Lin and others 2007). In the case of prolonged ER stress, apoptosis is initiated through activation of the apoptosis-mediating transcription factor CHOP, via the major UPR stress sensor pathways (reviewed by Colla 2019). CHOP-mediated apoptosis can occur as a result of its regulation of Bim and Bcl-2 (Puthalakath and others 2007) or through the dysregulation of protein synthesis machinery and oxidant stress in the ER (Marciniak and others 2004). When CHOP is turned on, it simultaneously up-regulates Bim, a key regulator of apoptosis, while suppressing Bcl-2, an antiapoptotic protein. Upon Bim activation, apoptosis is initiated as the mitochondrial pore forms, allowing for cytochrome C release and subsequent activation of caspase-9–mediated cleavage of caspase-3—a process normally inhibited by Bcl-2 (reviewed by Colla 2019).

The role of α-synuclein in disrupting ER proteostasis

The accumulation of α-syn is thought to cause cell death through a gain-of-toxic function, and it has been proposed that the buildup of aggregated proteins in the cell can overwhelm and sequester proteostasis factors such as chaperones, resulting in the deficient folding and processing of other proteins (reviewed by Sinnige and others 2020). Ultrastructural examination of the first α-syn transgenic mouse model indicated that electron-dense inclusions, possibly composed of α-syn, accumulate near the rough ER (Masliah and others 2000). Subsequent studies have shown that α-syn can colocalize and coimmunoprecipitate with GRP78 when overexpressed to high levels by plasmids in cell lines or in transgenic mice, leading to cell death by the ATF4 pathway (Bellucci and others 2011). Examination of A53T transgenic mice showed that they accumulate soluble SDS-resistant α-syn oligomers within the ER lumen (Colla and others 2012b). Analysis of the same mouse model during the course of disease progression showed that protein levels of GRP78 and GRP94 are not elevated in the presymptomatic stages but only increase when symptom onset and neuronal loss occur (Colla and others 2012a). Although this may indicate that the UPR was induced at late disease stages in vivo, the same study showed no changes in peIF2a/eIF2a ratio or ATF4-CHOP pathway occurrence, and mRNA levels of UPR chaperones were not reported. This result slightly contrasts with other studies (Bellucci and others 2011), which may be due to the levels of α-syn overexpression, the stage of pathology when the UPR was assessed, or the specific neuronal cell types that were examined in each study. Work from other groups also shows that α-syn associates with mitochondrial associated membranes of the ER (Guardia-Laguarta and others 2014; Paillusson and others 2017), specialized microdomains that mediate ER-mitochondrial contact sites and comprise cholesterol and anionic phospholipids. α-Syn preferentially binds anionic phospholipids with a particular curvature (Davidson and others 1998), and certain oxidized forms of cholesterol can potentiate α-syn aggregation (Bosco and others 2006). Therefore, it is possible that the pathogenic association of α-syn at ER membranes could be mediated by membrane curvature or budding of the ER membranes, as well as specific lipid composition of ER membranes. One concern with α-syn overexpression through artificial promoters is that changes in subcellular localization may occur from unnatural accumulation. However, studies that used iPSC models differentiated into midbrain dopamine neurons demonstrated that endogenously expressed α-syn accumulates into pathogenic species and also associates with ER chaperones CANX and GRP94, as well as membrane-fusing machinery of the early secretory pathway (Cuddy and others 2019; Mazzulli and others 2016; Stojkovska and others 2022). The disruption of ER homeostasis may occur via mislocalization of α-syn to the ER or early secretory pathway trafficking machinery that occurs upon its accumulation at the cell body (Mazzulli and others 2016). In addition to elevated α-syn levels, aggregation is specifically involved in perturbing trafficking of lysosomal hydrolases, given that overexpression of an aggregation-incompetent mutant, Δ71–82 α-syn, has no effect on lysosomal function (Mazzulli and others 2011, 2016). Aberrant association of aggregated α-syn with protein folding machinery could overwhelm and preoccupy chaperones, causing widespread misfolding of proteins.

Analysis of ER proteostasis in PD patient neurons has indicated that protein misfolding occurs within the ER at early stages in the pathologic cascade (Stojkovska and others 2022), along with the accumulation of certain ERAD substrates (Chung and others 2013; Cuddy and others 2019). In iPSC-midbrain models expressing A53T α-syn or SNCA-3X, maturity markers including extensive neuritic arbors and synapses are first detected at approximately 50 days after the start of differentiation using a standardized and highly reproducible protocol (Kriks and others 2011). Between days 60 and 90, soluble and insoluble α-syn species gradually accumulate, followed by neuron degeneration after day 110 (Cuddy and others 2019; Stojkovska and others 2022). In these models, protein misfolding of lysosomal beta-glucocerebrosidase (GCase) in the ER occurs at early stages of pathologic development when soluble and insoluble α-syn aggregates first become detectable but prior to neuron degeneration. Previous work found that immature forms of GCase accumulate in cells (Chung and others 2013; Gegg and others 2012; Mazzulli and others 2011). The importance of GCase in disease is further supported by the fact that an estimated 7% to 10% of PD patients have heterozygous loss-of-function pathogenic variants in GCase, qualifying it as the greatest genetic risk factor for PD and DLB (Chia and others 2021; Sidransky and others 2009). In healthy cells, GCase is synthesized in the ER and trafficked to the Golgi through lysosomal integral membrane protein 2 (LIMP-2) (Reczek and others 2007) (Fig. 2A,B), where it gets sorted into lysosomes. In PD, however, accumulation of α-syn in patient iPSC-derived neurons reduces GCase trafficking by both blocking membrane fusion at the cis-Golgi (Cuddy and others 2019) and decreasing ER proteostasis capacity (Stojkovska and others 2022). The buildup of immature GCase within the ER was found to be sufficient to trigger its conversion into insoluble aggregates (Fig. 2F), leading to downstream lysosomal dysfunction (Stojkovska and others 2022).

Figure 2.

Alternate handling of GCase in the endoplasmic reticulum (ER). (A) GCase is first translocated into the ER to be folded. (B) Under homeostatic conditions, properly folded GCase undergoes lysosomal integral membrane protein 2 (LIMP-2)–mediated vesicular transport to the Golgi and eventually to lysosomes. (C) If GCase misfolding occurs, ER chaperones (CANX/CALR) will assist in refolding. (D) If it remains misfolded, GCase will exit the folding cycle and is typically degraded by the proteasome via ER associated degradation (ERAD). (E) If ER stress persists, the unfolded protein response (UPR) is initiated. (F) In Parkinson’s disease patient neurons that harbor a wild-type SNCA triplication mutation, endogenous α-syn accumulation causes wild-type GCase to accumulate and form insoluble aggregates in the ER. Toxic GCase aggregation is insufficient to trigger the UPR in these neurons, despite their ability to respond to chemical UPR inducers. This suggests a deficiency in their ability to recognize and handle misfolded proteins in the ER. Further, misfolded wild-type GCase is not engaging with ERAD in these neurons. (G) ER-phagy is an additional quality control mechanism that selectively degrades regions of the ER via the autophagic pathway. Ongoing work aims to determine the contributions of ER-phagy in degrading ERAD-resistant cargo. Created with BioRender.com.

Normally, when cells accumulate misfolded proteins in the ER, the UPR is initiated to prevent insoluble aggregates from forming (Fig. 2E). A group of diseases exist that are characterized by pathologic protein aggregation in the ER, called ER storage diseases. These conditions are often caused by the expression of a mutant protein that induces rapid misfolding and aggregation into insoluble species so efficiently that it overwhelms chaperones and ERAD pathways of the UPR (reviewed by Li and Sun 2021). Perhaps the most well-studied ER storage disease is alpha1-antitrypsin deficiency, which causes cirrhosis through aggregation of mutated (Z variant) a1-antitrypsin. Here, inclusion bodies formed within fragmented ER structures exhibit toxic gain in function and form a molecular sieve in the ER, slowing the movement of other ER proteins in a size-dependent manner (Chambers and others 2022). The same study showed that ER chaperones such as CALR colocalize with antitrypsin polymers, which further stabilizes the aggregates and decreases chaperone mobility. In PD patient cultures, GCase accumulates within insoluble aggregates, although it is not known if these aggregates exhibit toxic gain in function as in the case of antitrypsin aggregation. A loss-of-function mechanism leading to cellular dysfunction has been suggested, since GCase aggregation prevented its mobility and trafficking into lysosomes (Stojkovska and others 2022). It is also clear that GCase is not the only protein that aggregates in the ER of PD cultures, since immature cathepsin D is also found as insoluble aggregates in synucleinopathy patient brain (Stojkovska and others 2022). The same study found that other hydrolases, like hexosaminidase B, remain soluble even though their immature forms accumulated in the ER. It is not clear what factors dictate the conversion of some but not all ER clients into insoluble aggregates, but it could be due to basic properties such as the level of protein expression, chaperone interactions, glycosylation status, the amount of time spent in the ER, or amino acid sequence that mediates protein solubility. Future studies should determine the mechanisms and specificity of the proteins that aggregate within the ER of PD patients.

It is currently unclear how misfolded proteins escape detection by the UPR in PD patient neurons, but it is possible that the GCase aggregates formed in the ER retain an ordered structure that is not recognized by UPR-sensing chaperones as “malfolded” per se. Aggregation of the WT form of GCase occurs in SNCA-3X iPSC-neuron models, and it is possible that aggregated GCase retained certain features of its native structure or did not exhibit sufficient exposure of hydrophobic patches that are required for GRP78 to permanently bind. This would allow minute amounts of degradation-resistant GCase aggregates to progressively accumulate over time, resulting in lysosomal dysfunction and propagation of proteostasis failure. Studies of Z-a1-antitrypsin aggregation provide a precedent for such a mechanism. These studies showed that polymerized aggregates of Z-a1-antitrypsin also do not induce UPR chaperone up-regulation, including GRP78 at early stages of accumulation, since its ordered structure is retained and not recognized as misfolded (Graham and others 1990). Therefore, it is possible that similar mechanisms occur in PD neurons. Detailed studies on the structure of GCase aggregates should provide further mechanistic insight into this process.

ER-localized α-syn can interact with ER chaperones that are involved in protein folding such as GRP78, GRP94, and CANX (Stojkovska and others 2022). This interaction could alter ER proteostasis by titrating these chaperones away from their normal folding clients, leading to the accumulation of unfolded substrates. While more studies are required to delineate the mechanism, it is possible that α-syn stabilizes the interactions of GRP78 with IRE1, ATF6, or PERK on the ER membrane, thereby preventing their downstream activation. Given that α-syn is a potent inhibitor of ER-Golgi trafficking, and ATF6 trafficking into the Golgi is required to form the active nuclear transcription factor ATF6(N) (Fig. 1), it is possible that α-syn could impede the UPR by preventing ATF6(N) formation (Credle and others 2015). Enhancing ER chaperone function through increasing ER-Ca2⁺ levels can improve GCase folding and solubility (Ong and others 2010; Stojkovska and others 2022), and overexpressing GRP78 in vivo rescues α-syn–induced toxicity (Gorbatyuk and others 2012), supporting the idea that ER chaperone function may be compromised by α-syn. Another observation that may explain the accumulation of misfolded aggregates is the dramatic morphologic changes that occur in the ER of PD patient neurons. In contrast to GD patient neurons that demonstrate ER expansion, the ER in PD neurons shrinks and fragments, despite the increased accumulation of immature proteins (Stojkovska and others 2022). ER fragmentation into vesicular inclusions occurs in other diseases such as a1-antitrypsin deficiency (reviewed by Carrell and Lomas 2002), as a result altered calcium (Koch and others 1988) or ER-phagy (see below). Calcium homeostasis has been shown to be disrupted in PD models (reviewed by Zaichick and others 2017) and therefore could play a role in disrupted ER morphology. ER morphology is controlled by structural proteins, including atlastins and reticulons (Orso and others 2009; Voeltz and others 2006), and it is possible that these structural proteins are deficient or dysfunctional in PD. Morphologic abnormalities and the failure to expand the ER compartment in PD neurons could promote protein aggregation through increasing local protein concentration in the absence of chaperone up-regulation. A reduction in ER area could occur through faulty XBP1 signaling, which functions to stimulate phospholipid synthesis genes that build the ER membrane (Schuck and others 2009; reviewed by Walter and Ron 2011). Interestingly, changes in phospholipid content have been documented in cell culture models of PD (reviewed by Fanning and others 2020). These changes may occur as a cause or consequence of the ER fragmentation phenotype, given that the ER is a central location of phospholipid synthesis in the cell.

By comparison to PD neurons, in GD neurons that express mutant GCase, it is clear that the UPR is successfully activated and efficiently clears the mutant protein, leading to near-complete depletion of GCase (Lang and others 2019; Ron and Horowitz 2005; Stojkovska and others 2022). EDEM levels are elevated in GD neurons, and GCase mutant protein responds to proteasomal inhibition (Ron and Horowitz 2005; Stojkovska and others 2022), suggesting that ERAD plays a role in its clearance. Similarly, PD patient neurons that endogenously express a GBA1 mutation show signs of ER stress, documented by up-regulated UPR chaperones at the protein level (Fernandes and others 2016). When mutant forms of GCase are ectopically expressed in PD SNCA-3X neurons by lentivirus, the UPR fails to respond (Stojkovska and others 2022) (Fig. 2F). The same study found that proteasomal block in SNCA-3X neurons had no effect on accumulated WT GCase, suggesting that despite the need for its clearance, GCase is not degraded via ERAD. Further, α-syn levels and EDEM1 protein levels were negatively correlated in these neurons, pointing to α-syn–induced ERAD dysfunction. Together, this is suggestive of a deficiency in either the UPR sensors or transducers in PD neurons, the mechanisms of which have yet to be determined. Given that GD neurons also accumulate α-syn, and studies in isogenic PD models show that α-syn is responsible for blunting the UPR response, it is not clear how GD neurons can trigger the UPR. It is possible that the UPR dysfunction only occurs once a critical threshold of α-syn accumulation occurs, since SNCA-3X patient neurons accumulate more α-syn compared to GD neurons.

Although PD neurons at early pathologic stages did not demonstrate UPR activation in the presence of misfolded proteins, stimulation with chemical ER stressors, including thapsigargin or brefeldin A, could successfully induce the UPR (Stojkovska and others 2022). This suggests that a specific trigger induced by protein misfolding, but not through chemical stimulation, is perturbed. Recent studies have shown that chemical induction of the UPR is distinct from protein misfolding stress, producing pleiotropic activation of multiple cellular stress pathways (reviewed by Bergmann and Molinari 2018). It is possible that α-syn blunts the UPR machinery required for sensing misfolded proteins and therefore requires a more dramatic stress stimulus that induces stress beyond that of protein misfolding to initiate the transcriptional response. Chemical induction in addition to α-syn–induced stress may be sufficient to trigger the response by surpassing this threshold.

Other studies have documented a connection between ER stress, the UPR, and cell death in postmortem PD brain, culture models, or mouse models that accumulate pathogenic α-syn. For example, one study reported ER chaperone accumulation at the protein level within Lewy bodies (Conn and others 2004). Further, an up-regulation of the PERK UPR pathway was reported in dopaminergic neurons in postmortem patient tissue (Hoozemans and others 2007). This study analyzed 13 PD brains and detected phospho-PERK or peIF2a proteins by immunohistochemistry in 1% to 15% of neurons. Other studies in both transgenic overexpression synucleinopathy models have suggested UPR initiation that corresponds to late-stage pathology. Colla and others found that in aged A53T transgenic mice (A53TαS Tg), UPR induction, detected at the protein level by grp78 and grp94 immunohistochemistry, selectively occurs in neurons that show α-syn pathology (Colla and others 2012a). Further, studies in iPSC-derived cortical neurons that harbor SNCA-3X reported “protein processing in the ER” as a top category along with other categories, including apoptosis, identified by unbiased transcriptomic analysis (Heman-Ackah and others 2017). Other studies in early-stage iPSC midbrain neurons, which we define as cultures that exhibit markers of neuronal maturity and first-detectable insoluble α-syn, showed that while GRP78, GRP94, and calnexin protein levels were mildly elevated, the mRNA levels of these chaperones were decreased, suggesting that the UPR was not activated (Stojkovska and others 2022). Since the UPR initiates as a transcriptional response, it can be more accurately assessed by measurements of mRNA of UPR-responsive elements, as opposed to protein levels that can change because of altered protein clearance. It is important to note that some studies that claim activation of the UPR only measure protein levels but do not assess the transcriptional response, which may explain some of the conflicting data in the literature. Nevertheless, findings from several laboratories support the idea that ER stress occurs in PD and that UPR markers are detectable at late-stage disease after detectable neuron degeneration occurs.

Many in vivo and in vitro studies examining mechanisms of PD pathogenesis have implicated ER stress. Early studies in tyrosine hydroxylase expressing neuroblastoma cells found that treatment with 6-OHDA and MPP⁺, neurotoxins that induce PD-like phenotypes, results in the transcriptional up-regulation of UPR genes (Holtz and O’Malley 2003). Experimental in vivo systems in mice found that CHOP deletion was protective against neurotoxins in dopaminergic neurons, including 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Silva and others 2005), although it is not clear if these chemical toxins involve α-syn. Some studies have also shown that dopaminergic neurons are selectively vulnerable to ER stress. One study in adult mice found that global silencing of XBP1 causes chronic ER stress and selective degeneration of dopaminergic neurons in the substantia nigra. In-terestingly, they found local delivery of XBP1 via gene therapy to be protective against parkinsonism-inducing neurotoxins (Valdes and others 2014).

ER-phagy

In addition to ERAD and the UPR, recent work indicates that ER homeostasis is maintained by the autophagic pathway, in a selective process termed ER-phagy (Fig. 2G). Here, regions of the ER are pinched off and engulfed by autophagosomes in a ubiquitin-independent manner (reviewed by Grumati and others 2018). Multiple ER-resident proteins have been identified as regulators of ER-phagy. FAM134B, the first identified ER-phagy receptor, regulates selective ER degradation through its clustering in the ER membrane in response to local stress. Upon clustering, FAM134B interacts with the essential autophagic protein LC3 during the initial stages of autophagosome formation. The clustering of these proteins concentrates LC3 interacting regions (LIRs) in the ER membrane as well as reshapes the membrane itself, providing the driving force to begin the shedding of a membrane or vesicle (Khaminets and others 2015). The identified ER-phagy receptors, including CCPG1 (Smith and Wilkinson 2018), ATL3 (Chen and others 2019), RTN3L (Grumati and others 2017), SEC62 (Fumagalli and others 2016), and TEX264 (Chino and others 2019), differ in the number of LC3 molecules they can bind, their association with autophagy initiation machinery, and propensity to induce ER membrane curvature. While these differences slightly alter the way ER-phagy is initiated, once the ER is engulfed by autophagosomes, ER-phagy progresses in the same way as other forms of autophagy, where autophagosomes fuse with lysosomes and hydrolases break down cargo (reviewed by Wilkinson 2020).

Ongoing research in the ER-phagy field tries to resolve how certain subdomains of the ER are cleared while others are left behind. A growing consensus is that both transmembrane and luminal ER chaperones function to transduce local ER stress to cytosolic autophagy initiation machinery by simultaneously binding both misfolded proteins in the ER lumen and ER-phagy receptors themselves. This effectively clusters ER-phagy receptors within the ER membrane, enabling multivalent interactions with the initial isolation membrane that forms autophagosomes (phagophore) and pinching off from the site of local ER stress and delivering the damaged components to auto-lysosomes for degradation (reviewed by Wilkinson 2020).

Recent work demonstrated that misfolded proteins can be cleared by ER-phagy via CANX-FAM134B interactions (Forrester and others 2019). As mentioned above, α-syn aberrantly interacts with CANX in SNCA-3X neurons and contributes to misfolded GCase in the ER (Stojkovska and others 2022). Moreover, the ER fragmentation observed in these neurons is reminiscent of that observed upon FAM134B overexpression both in vitro and in vivo (Khaminets and others 2015; Stojkovska and others 2022). Genetic manipulation of FAM134B in human cells and mice is sufficient to induce ER morphologic changes and modulate ER turnover (Khaminets and others 2015). This demonstrates the critical contributions of ER-phagy to ER homeostasis and raises the possibility that α-syn impairs ER-phagy through aberrant interaction with ER chaperones, ER-phagy receptors, and possibly disrupting their interactions with each other (Fig. 3A). Additionally, α-syn has an affinity for charged curved membranes (Pranke and others 2011). While the ER and ER-vesicles may be larger than what physiologic α-syn has been observed to bind to (i.e., synaptic vesicles), oligomeric or aggregated forms of α-syn could bind to curved or uncurved portions of ER membranes. Indeed, previous work showed that subtypes of toxic α-syn oligomers bind and disrupt membranes (Fusco and others 2017) and are directly associated with ER-derived membranes and microsomes (Colla and others 2012a, 2012b, 2018). Therefore, it remains possible that the affinity of α-syn for ER membranes could disrupt the interaction between ER-phagy receptors and autophagosomes (Fig. 3B). Overall, the mechanisms leading to ER proteostasis failure in PD are not understood. It may be possible that disrupted ER-phagy causes an imbalance in ER homeostasis leading to downstream dysfunction of the ALP in PD, uncovering a novel pathogenic mechanism and offering therapeutic targets that restore proteostasis.

Figure 3.

Potential α-synuclein (α-syn) interference mechanisms of ER-phagy. (A) α-Syn has been reported to associate with endoplasmic reticulum (ER) chaperones that assist in ER quality control and ER-phagy (CANX, GRP78, GRP94). This may result in reduced chaperone output during ER stress and impair selective ER turnover. (B) Given its affinity for charged, curved membranes, it is possible that α-syn disrupts ER-phagy receptor-autophagic machinery interactions via LC3-interacting regions (LIRs) on the receptor and LC3 molecules on the growing phagophore. This could disrupt the proper turnover of select cargo and the ER membrane upstream of autophagosome-lysosome fusion. (C) α-Syn impairs late-stage autophagy through blockage of autophagosome-lysosome fusion. This occurs via aberrant interaction with the SNARE protein ykt6. Created with Biorender.com.

ER-Golgi trafficking

Following the synthesis, folding, and processing in the ER, proteins must be sorted and packaged before reaching their destination. These integral processes occur in the Golgi. However, before a protein in its native conformation can be sorted, it must exit the ER and be successfully delivered to the Golgi. The trafficking events that occur between these two organelles rely on vesicular transport and the tight regulation of the budding and fusing of membranes. In brief, vesicles emerge from specialized coated areas of a membrane. These coats contain adaptor proteins that are essential for packaging transmembrane proteins and their respective cargo inside a transport vesicle. Prior to fusion with a target membrane, the vesicle coat is shed. Rab GTPases are key regulators of vesicle budding and transport while SNARE proteins mediate fusion with target membranes (reviewed by McCaughey and Stephens 2019).

During ER-Golgi trafficking, properly folded proteins exit the ER in COPII-coated vesicles that bud off from regions of the ER without ribosomes known as exit sites. Like the principles of ER-phagy initiation, the clustering of membrane proteins that contain cytosolic exit signals recruits COPII adaptor proteins, thus promoting vesicle formation. The vesicle then sheds its coat and fuses with nearby vesicles to form tubular clusters. These vesicle clusters travel on Golgi-bound microtubule tracks, promoting their fusion with each other and the formation of the cis-Golgi network (reviewed by McCaughey and Stephens 2019). Specifically, the programming of COPII vesicles for fusion relies on the interaction between Rab GTPases, like Rab1a, and vesicle docking proteins, like p115. This interaction promotes the association with the Golgi membrane through cis-Golgi matrix protein GM130, which provides an adherent surface to promote membrane fusion. Once a cognate SNARE partner is found, membrane fusion occurs (reviewed by Wang and others 2020). Membrane trafficking occurs in both forward and reverse directions to achieve balance in trafficking machinery. For example, there are retrieval pathways in place that use sorting signals to deliver misplaced cargo, usually ER resident proteins, back to the ER. The back-and-forth traffic of vesicles between the ER and Golgi is critical in ensuring proper protein delivery and ultimately function (reviewed by McCaughey and Stephens 2019).

Disruptions in ER-Golgi trafficking can lead to aberrant secretion of nonfunctional misfolded proteins or, as discussed above, the accumulation of misfolded proteins that accumulate in aberrant locations. The importance of ER-Golgi protein trafficking on lysosomal and neuronal function is best exemplified by a rare neuronopathic lysosomal storage disorder called inclusion-cell disease (I-cell), or mucolipidosis type II (MLII). In I-cell disease, a loss-of-function mutation in the Golgi enzyme N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (encoded by GNPTAB) results in loss of M6P signal that is required for lysosomal transport. Without the M6P modification, lysosomal enzymes are secreted from cells instead of reaching their final destination of lysosomes (Hickman and Neufeld 1972). Since most lysosomal enzymes depend on M6P, apart from acid phosphatase and GCase, this results in dysfunctional lysosomes that accumulate multiple protein, lipid, and glycan substrates and appear as inclusions in patient cultured cells. GNPTAB deficiency is characterized by severe degeneration of the nervous system in humans and in mouse models (Kollmann and others 2012), indicating the importance of proper trafficking and lysosomal function particularly for neurons.

In the case of α-syn accumulation, multiple studies have shown that trafficking between the ER and Golgi is compromised and severe Golgi fragmentation occurs upon α-syn overexpression (Cooper and others 2006; Gitler and others 2008; Gosavi 2002 and others; Thayanidhi and others 2010). It has been previously reported that SNARE protein function is sensitive to both overexpressed and endogenous α-syn accumulation as it can directly bind ER-Golgi SNARE proteins, preventing SNARE complex assembly (Cuddy and others 2019; Thayanidhi and others 2010). Specifically, α-syn can disrupt ykt6 function, a key SNARE protein involved in ER-Golgi trafficking (Fukasawa and others 2004; Liu and Barlowe 2002; McNew and others 1997; Zhang and Hong 2001), through aberrant binding. This prevents ykt6 from contributing to the lysosomal stress response through supporting hydrolase trafficking (Cuddy and others 2019). Interestingly, knockdown of ykt6 in human midbrain neurons results in preferential reduction of lysosomal machinery and depletion of lysosomal compartments. Studies showed that ykt6 becomes active upon lysosomal inhibition and coordinates with the master regulator of lysosomal biogenesis, transcription factor EB (TFEB) (Cuddy and others 2019). The trafficking of lysosomal hydrolases could be rescued by overexpression of ykt6, but other proteins that mature through the secretory pathway, including secreted and plasma membrane proteins, were not affected by ykt6. This indicates that ykt6 may play a preferential role in lysosomal biogenesis through enhancing the trafficking of hydrolases. Therefore, it seems clear from multiple studies that impaired membrane fusion events are a major phenotype in α-syn–expressing cells. In animal models of PD, overexpression of Rab1 and other components of ER-Golgi transport was found to be protective against α-syn toxicity in dopaminergic neurons (Cooper and others 2006). Consistent with this finding, α-syn accumulation interferes with Rab1 ER-Golgi localization in PD patient cultures. These neurons show increased trafficking and activity of lysosomal hydrolases and reduced α-syn levels upon Rab1 overexpression (Mazzulli and others 2016). Together, this demonstrates the importance of ER-Golgi protein transport to lysosomal function and neuronal health. Further, these findings support a major role for protein trafficking in PD pathogenesis and provides insights into the mechanisms of α-syn toxicity.

Golgi and post-Golgi trafficking

In addition to being a hub for the synthesis of carbohydrates, the Golgi serves as a sorting site for proteins that leave the ER. To be properly targeted for either secretion or transport to organelles, proteins require post-translational modification in its cisternae. Here, proteins are sorted and tagged by oligosaccharides directing their trafficking. In the cis-Golgi, lysosome hydrolase precursor proteins that are destined for lysosomes get tagged by M6P moieties. Proteins with the M6P targeting signal progress to the trans-Golgi network (TGN), a highly dynamic structure of protruding tubules at the end of the Golgi complex that serves as an important organizational interface for targeting proteins to the plasma membrane, early and late endosomes, recycling endosomes, and other specialized organelles such as the lysosome (reviewed by De Matteis and Luini 2008). There, they are bound by the M6P receptor and progress in a complex to late endosomes. The low pH of this compartment causes the dissociation of the receptor from the precursor protein, and upon fusion of late endosomes with lysosomes, the hydrolase is delivered (reviewed by Fullekrug and Nilsson 1998). This pathway is often exploited when treating LSDs via enzyme replacement therapy (reviewed by Coutinho and others 2012). Additionally, some lysosomal hydrolases are delivered via an M6P-independent pathway. GCase is one such enzyme, which relies on its interaction with LIMP-2 for delivery to lysosomes. In LIMP-2 knockout cells, GCase is aberrantly trafficked resulting in its secretion out of the cell instead of delivery to lysosomes (Reczek and others 2007). It is important to note that despite trafficking deficits in LIMP2^–/– mice, GCase is not retained in the ER and does not aggregate (Stojkovska and others 2022), as it does in SNCA-3X PD patient neurons. This suggests that the GCase aggregation phenotype is selective for PD neurons. Studies in LIMP-2 knockout mice have shown that α-syn accumulates into pathogenic inclusions similar to what is observed by GCase depletion (Rothaug and others 2014). LIMP-2 mutations in humans cause action myoclonus renal failure syndrome, a rare disease characterized by myoclonic epilepsy, ataxia, and the accumulation of storage material in the brain, leading to neurodegeneration. This demonstrates the critical importance of LIMP-2 in proper GCase trafficking and lysosomal function of neurons.

The sorting of proteins through the TGN is essential for neuronal homeostasis (reviewed by Valenzuela and Perez 2015). α-Syn–induced disruptions to the early secretory pathway, which includes Golgi homeostasis, have been extensively implicated in disease pathogenesis (reviewed by Wang and Hay 2015). α-Syn itself is associated with components of the ER-Golgi-TGN biosynthetic pathway, and dysregulated α-syn levels alters the function and morphology of the Golgi and TGN (Chutna and others 2014; Fujimoto and others 2018; Gosavi and others 2002; Huber and others 1993; Tomás 2021nisms of α-syn accumulation in cells revealed dysfunction in the early secretory pathway, characterized by severe Golgi fragmentation. This phenotype occurred in cells that accumulated prefibrillar α-syn species and was associated with a decline in the maturation and trafficking of the dopamine transporter (Gosavi and others 2002). Studies in PD patient SNCA-3X midbrain neurons also showed dramatic Golgi fragmentation and colocalization of α-syn with cis-Golgi markers, including GM130 (Mazzulli and others 2016). More recent histopathologic evidence similarly showed altered Golgi morphology in the surviving dopaminergic neurons of postmortem PD substantia nigra pars compacta (SNc) tissue that was correlated with elevated levels of Rab1 and Golgin-84 (Tomás and others 2021), a Golgi matrix protein required for stack assembly and cisternae lengthening, as well as a Rab1 binding partner (Satoh and others 2003). Further, a recent study in rodent midbrain cultures reported increased Golgi fragmentation, as well as decreased neurite length and increased fission with mitochondria, associated with A53T overexpression. Interestingly, they found that the effect was greater with additional knockdown of putative kinase 1 (PINK1), an important regulator of mitochondrial health whose variants are causal for early-onset PD, pointing to a possible mechanistic convergence (Furlong and others 2020). While the cause/effect association of α-syn accumulation and Golgi fragmentation is currently unclear, the higher proportion of fragmented Golgi in cells containing mutant or prefibrillar α-syn (versus fully formed fibrils) suggests a possible causal role for α-syn in the dispersed Golgi morphology early in PD pathogenesis.

Evidence for α-syn–induced disruptions of the TGN is more direct. α-Syn has been shown to interact with Rab8a (Yin and others 2014), a GTPase involved in regulating transport from the TGN to the plasma membrane (Huber and others 1993). Overexpression of Rab8a in two Drosophila models of α-syn toxicity mitigated the development of locomotor impairment (Yin and others 2014). Additionally, Rab11, a GTPase that, among other functions, regulates TGN trafficking, colocalizes with α-syn and modulates its aggregation (Chutna and others 2014). In HEK cells containing a hyperactive LRRK2 mutant associated with the accumulation of α-syn and the pathogenesis of PD, the increased phosphorylation of Rab7L1 was associated with a more diffuse TGN morphology, although the functional significance is unclear (Fujimoto and others 2018). Further, α-syn can impair the movement of hydrolases from the TGN to lysosomes via the M6P signaling pathway—a reduction in the M6P receptor was observed in cell lines and transgenic mouse models overexpressing WT α-syn, as well as postmortem PD brain tissue (Matrone and others 2016). A reduction in the M6P receptor has been correlated with reduced levels of the key lysosomal protease cathepsin D. Cathepsin D is ubiquitously expressed throughout the body but is enriched in the brain, and impairment in its function or reduced expression is associated with the pathogenesis of PD (Bunk and others 2021). Given that early secretory pathway dysfunction is a hallmark of PD, the relationship between α-syn and Golgi function and morphology is a growing area of interest to better understand disease mechanisms.

Endolysosomal pathway and α-synuclein

The endolysosomal system is necessary for maintaining proteostasis, cell signaling, and survival. This tightly controlled pathway regulates the recycling and degradation of membrane-bound proteins, endocytosed extracellular material, and proteins synthesized and matured through ER-Golgi trafficking (reviewed by Teixeira and others 2021). Highlighting the relevance of this pathway to PD, several genes, including LRRK2, SNCA, DNAJC6, SYNJ1, GAK, SH3GL2, VPS35, and RME-8, are associated with an elevated risk for PD (Nalls and others 2019). Among the regulatory proteins mediating vesicle trafficking within the endolysosomal system are the family of small Rab GTPases, Endosomal Complex Required for Transport (ESCRT 0, I, II, and III), and several SNAREs. The functions of these proteins and protein complexes can be disrupted by the overexpression or aggregation of α-syn, forming a positive feedback loop through which their impairment can lead to further α-syn accumulation and neurotoxicity (Nemani and others 2010; reviewed by Shi and others 2017; Spencer and others 2016; reviewed by Teixeira and others 2021). The protein encoded by LRRK2, a familial PD gene, is a key regulator of the endolysosomal system (Paisán-Ruíz and others 2004). LRRK2 is a large, multidomain protein that displays both kinase and GTPase enzymatic activity in addition to containing several protein interaction and scaffolding domains. Despite its low expression in neurons, LRRK2 loss of function reduces protein degradation and results in α-syn accumulation in vivo (Tong and others 2010). The most common Parkinson’s linked variant in LRRK2 is a G2019S missense mutation in its kinase activation loop that increases phosphorylation of a subset of Rab GTPases (reviewed by Hur 2019). For example, LRRK2 can decrease late endosome fusion with lysosomes through hyperphosphorylation of Rab7 (Gómez-Suaga and others 2012). Given that LRRK2 is capable of performing numerous cellular functions, the effect of its hyperactivity is diverse, but particular focus has been placed on the deleterious effects on the endolysosomal system and the resulting α-syn aggregation (reviewed by Erb and Moore 2020).

Autophagic-lysosomal pathway and α-synuclein

Importantly, disposal of damaged proteins in the cytosol is not limited to the proteasome. Similarly to protein degradation processes originating in the ER, where both ERAD (proteasome) and ER-phagy (the lysosomal equivalent) work concomitantly, the same holds true for cargo slated for degradation in the cytosol. In addition to the UPS, macroautophagy, which we refer to as autophagy, is a critical portion of the protein homeostasis network regulating the clearance of misfolded proteins and damaged organelles. This process relies on the coordinated activity of autophagy-related (Atg) proteins that mediate the sequestration of damaged proteins and organelles in double-membrane vesicles called autophagosomes, which fuse with lysosomes where their contents are degraded (reviewed by Menzies and others 2017). Simply, autophagy is triggered by the formation of the initiation complex, helmed by the protein ULK1, and its recruitment to the ER membrane for cleavage (where autophagosome membranes are derived) (reviewed by Nakatogawa 2020). ULK1 is a key regulator of autophagy—its activity depends heavily on the physiologic environment. For example, in a nutrient-rich environment, ULK1 is phosphorylated by the nutrient-sensor mTOR, and its kinase activity is inhibited. Upon mTOR inhibition (e.g., in the context of starvation where energy ratios are altered), the ULK1 is free, allowing for the formation of the initiation complex and autophagosome biogenesis (reviewed by Szwed and others 2021). Following initiation, nucleation occurs with the assembly of the PI3K complex, helmed by the protein Beclin1. This complex facilitates membrane elongation and phagophore formation. Once the phagophore membrane is isolated, the important autophagy regulatory protein LC3 is converted to LC3II (reviewed by Tanida and others 2004), resulting in an autophagosome vesicle ready for fusion with the lysosome.

Neuron-specific deletion of essential autophagy genes in mice results in neurodegeneration (Ahmed and others 2012; Friedman and others 2012; Komatsu and others 2006; reviewed by Menzies and others 2017), highlighting the importance of autophagy within the nervous system. In addition to the histopathologic evidence of α-syn accumulation in PD brains, genetics have clearly shown that dysfunction of the ALP plays an important role in PD etiology. Several PD risk genes have been identified in patient genome-wide association studies (GWAS) that are critical for lysosomal function and autophagy, including GPNMB, TMEM175, and, most notably and discussed previously, GBA1 (reviewed by Aflaki and others 2017; Chang and others 2017; Nalls and others 2014). Loss-of-function mutations in GCase, a glycosphingolipid hydrolase, directly induce α-syn aggregation by interaction with sphingolipid substrates inside lysosomes (Fredriksen and others 2021; Mazzulli and others 2011; Zunke and others 2018). Interestingly, even in the absence of ALP-implicated variants, α-syn can inhibit the maturation of lysosomal hydrolases (Mazzulli and others 2011, 2016) and interact with degradation machinery directly (Cuervo and others 2004; Freeman and others 2013; Yap and others 2013), resulting in ALP dysfunction. Moreover, in vivo work has shown that α-syn overexpression induced autophagic defects in mice (Chen and others 2015). Taken together, this demonstrates that even beyond GBA1, there is strong genetic and biochemical evidence linking loss of lysosomal activity and autophagic trafficking components in α-syn toxicity and PD pathogenesis.

Understanding the exact toxic mechanisms α-syn in autophagy is of high interest. To date, we know α-syn is capable of disrupting autophagy at several steps, including both autophagosome formation and autophagosome-lysosome fusion (Tang and others 2021; Winslow and Rubinsztein 2011). It is hypothesized that autophagosome formation is disrupted in a Rab1a-dependent mechanism (Winslow and Rubinsztein 2011). The fusion of an autophagosome with a lysosome is the culminating step of autophagy, and the process is carefully controlled through the involvement small Rab GTPases, SNAREs, and tethering proteins (reviewed by Lorincz and Juhasz 2020). The accumulation of autophagosomes in PD nigral tissue has been long documented (Anglade and others 1997), suggesting that autophagic flux is impaired at this final step. SNAP29 is a necessary SNARE involved in several membrane-fusion events, including autophagosome-lysosome fusion (reviewed by Tian and others 2021). The overexpression of α-syn in cultured cell lines reduces the abundance of SNAP29 and reduced autophagosome-lysosome fusion (Tang and others 2021). These cells show an increase in extracellular vesicle–related proteins in the culture medium to similar levels, potentially suggesting a compensatory extracellular secretion of autophagy substrates to reduce proteasomal load. The same study also showed a stepwise reduction in SNAP29 from Braak stages 1 to 3 in the SNc of PD and DLB patients, consistent with the hypothesis that increasing levels of α-syn could reduce SNAP29 levels (Tang and other 2021). Tangentially, another recent study found LRRK2 variants with hyperactive kinase functions could potentially block autophagosome-lysosome fusion, as it was shown that the beneficial effect of a LRRK2-kinase inhibitor was reliant on this final step in the pathway (Obergasteiger and others 2020).

We recently showed that autophagy is also inhibited at autophagosome-lysosome fusion in familial SNCA-3X iPSC patient neurons that accumulate α-syn through endogenous overexpression (Pitcairn and others 2023) (Fig. 3C). While autophagosome markers LC3-II and p62 were increased at baseline compared to CRISPR-corrected controls, flux assessed by lysosomal inhibitor treatment was reduced, indicating a late-stage blockade. More directly, α-syn accumulation decreased colocalization between autophagosomes and lysosomes, visualized in fixed cells. Just as pathogenic α-syn disrupts ykt6 SNARE complexes required for its hydrolase trafficking activity, we found that ykt6-SNAP29 complexes mediating autolysosome fusion were also reduced, although total SNAP29 protein abundance appeared unchanged. Ykt6 knockdown in healthy neurons recapitulated flux deficits, and autophagy in PD neurons could be rescued by ykt6 overexpression or pharmacologic activation (Pitcairn and others 2023). Given previous findings (Cuddy and others 2019), ykt6 thus appears vital for coordinating the delivery of lysosomal enzymes with the arrival of autophagy substrates in response to cellular stress. Some of α-syn’s characteristic neurotoxicity may be explained by its ability to interfere with a protein required for two arms of this pathway, making ykt6 a potential target for therapeutic intervention.

Further support for the relationship of ALP dysfunction and α-syn toxicity comes from early work investigating chaperone-mediated autophagy (CMA) in PD. Briefly, CMA differs from macroautophagy in that it does not use autophagosomes. Instead, CMA requires the recognition of amino acid motifs by the cytosolic chaperone HSC70 that then targets proteins to the lysosome and mediates engulfment through a lysosomal transmembrane protein, LAMP-2A, dependent mechanism (reviewed by Kaushik and Cuervo 2018). Interestingly, α-syn mutations that lead to early-onset PD (A53T and A30P) were bound with CMA receptors and blocked uptake into the lysosome (Cuervo and others 2004). A similar effect was observed by dopamine modified α-syn (Martinez-Vicente and others 2008). Recent work indicates mutant GCase may also be a CMA antagonist, reporting its recognition by HSC70 and subsequent clustering around lysosomes, blocking LAMP2A recycling and α-syn degradation by CMA (Kuo and others 2022).

Concluding remarks

In PD, it is well established that α-syn disrupts multiple branches of the proteostasis pathway, including protein trafficking. α-Syn–induced perturbations to the trafficking of lysosomal proteins between the ER and Golgi have been implicated in multiple PD models, from yeast to patient-derived neurons. Interestingly, recent data indicate that disrupted ER homeostasis also plays a critical part in pathogenesis (Fig. 4). The relative contributions of a faulty ER stress response or ER degradative pathways are not completely understood. Autophagy plays a critical role in maintaining cellular health, and genetic evidence shows a clear involvement of the ALP in PD. While ER-phagy is a known conserved cellular process for maintaining ER homeostasis via selective autophagy, little is known about its contributions to the ER phenotypes commonly seen in synucleinopathies. A deeper understanding of the relationship between pathogenic α-syn accumulation, ER-phagy, and other ER degradative mechanisms may advance the field in identifying targetable pathways.

Figure 4.

Intracellular protein trafficking phenotypes in α-synuclein (α-syn) accumulation Parkinson’s disease models. Accumulation of α-syn intracellularly is associated with proteostatic change at multiple stages of secretory trafficking. This includes endoplasmic reticulum (ER) function, morphology, and quality control mechanisms; ER-Golgi trafficking; Golgi and trans-Golgi network function and morphology; hydrolase trafficking and lysosomal function; the endolysosomal pathway; and the autophagic-lysosomal pathway. Created with BioRender.com.

PD is among the neurodegenerative diseases whose pathogenesis is linked to the toxic accumulation of a hallmark protein. An ongoing question in the field is, what makes a particular protein toxic to a cell? In the case of protein trafficking, one possible explanation is the inherent physical properties of α-syn. In addition to its nonamyloid component domain that renders the protein aggregation prone, α-syn has an N-terminal amphipathic region that contains seven KTKEGV repeats (Maroteaux and others 1988)—a motif that allows it to interact with membranes (Burré and others 2014; Davidson and others 1998; Theillet and others 2016), suggesting its ability to interfere with the movement of proteins through various membrane structures either through direct interactions or by overwhelming transport machinery.

Currently, there are no disease-modifying therapies for the treatment of any synucleinopathy. Looking forward, the development of trafficking-enhancing therapies offers promise as a method to clear the toxic accumulation of α-syn. Given well-established genetic and biochemical data, there is a case to be made that impairments in trafficking are driving disease. For most PD cases, there is no single disease-causing mutation. However, genetic studies in idiopathic patients point to a possible polygenic effect of known trafficking genes—in particular, those converging on the autophagic-lysosomal pathway. While the application of gene editing in human disease is still fledgling, pharmacologic enhancement of autophagy has strong therapeutic potential for PD and multiple other neurodegenerative diseases, including Alzheimer’s disease (Majumder and others 2011), Huntington’s disease (Carmichael and others 2002; Sarkar and others 2009; Tanaka and others 2004), and amyotrophic lateral sclerosis (Fornai and others 2008). Regardless, it is likely that treatments targeting multiple branches of the trafficking pathway are likely needed. Recent work describes a synergistic approach targeting both chaperone function and downstream ER-Golgi trafficking, which rescued hydrolase maturation and lysosomal function, improved ER morphology, and reduced α-syn in SNCA-3X patient-derived neurons (Stojkovska and others 2022). Recent studies have also described the protective power of UPR activation against neurodegeneration (Grandjean and others 2020; Vidal and others 2021), suggesting enhancement of this pathway has therapeutic potential. Overall, intracellular protein trafficking is critical for maintaining cellular health, and its dysfunction is a hallmark of PD. Current and future translational research will continue to uncover the underlying mechanisms of trafficking deficits and α-syn toxicity with the overarching goal of improving disease outcomes.