The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

␣-Synuclein (␣syn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). ␣syn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous ␣syn. The cell-to-cell release and uptake of ␣syn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in ␣syn spread has yet to be characterized. In this study, we used a paracrine ␣syn in vitro neuronal model to test the impact of Rab27b on ␣syn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased ␣syn-mediated paracrine toxicity. Rab27b reduced ␣syn release primarily through nonexosomal pathways, but the ␣syn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble ␣syn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of ␣syn and, ultimately, in the pathogenesis of synucleinopathies.
Synucleinopathies, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), have tremendous economic and social impact, yet there are no current therapies to slow neurodegeneration in PD or DLB (1). ␣-Synuclein (␣syn) is the key pathogenic protein implicated in both PD and DLB. Recent evidence suggests that misfolded ␣syn propagates from neuron to neuron in a prion-like manner: transmission of misfolded ␣syn templates misfolding of endogenous ␣syn to promote further aggregation and ultimately neurotoxicity (2)(3)(4). Propagation of ␣syn requires three distinct cellular processes: release, uptake, and misfolding. ␣syn does not have a signal peptide and is not released via the classic endoplasmic reticulum-Golgi secretory pathway (5,6). There is evidence that ␣syn is released through nonclassical endosomal pathways, including exosomal, misfolding-associated protein secretion, and lysosomal pathways (7)(8)(9)(10)(11)(12)(13). These pathways are thought to be tightly regulated by the Rab family of GTPase proteins.
Rab GTPases are a largely conserved family of proteins with over 60 known mammalian members. Rab proteins' primary functions are carried out through a catalytic GTP/GDP binding site, which, when GTP-bound, causes a conformational switch allowing the protein to interact with its effector proteins and thus regulate multiple cellular processes (14). Several Rab GTPases have been associated with PD. Mutations in Rab39b and Rab32 cause autosomal recessive forms of early-onset and late-onset PD, respectively (15)(16)(17). Rabs regulate autophagic, exocytotic, and endocytotic pathways potentially involved in ␣syn transmission. A number of Rab GTPases regulate ␣syn trafficking (18). Overexpression of Rab1, -7, -8, or -11 is protective against ␣syn toxicity, and these Rabs are implicated in ␣syn aggregate formation in PD models (19 -24). Additionally, Rabs have been shown to be kinase substrates of LRRK2, mutations of which are the most common genetic cause of PD (25).
Rab27b is a relatively unstudied member of the Rab GTPase family. Pointing to a potential functional overlap between the two proteins, Rab27b shares common regulators and effectors with Rab3, which binds ␣syn and is localized in ␣syn aggregates (14,22,(26)(27)(28). Rab27b can regulate protein secretion through both exosomal and nonexosomal pathways by regulating transport and docking steps in tandem with Rab27a through interaction with several Rab27 effectors (29 -35). Rab27b and its effectors regulate exocytosis of dense-core vesicles in neuronal lines and synaptic vesicle release in neurons (27,28,(36)(37)(38). Unlike Rab27a, Rab27b is highly expressed in neurons in the cortex, striatum, and midbrain, areas affected in PD and DLB (29,39).
Rab27b may also play a role in protein clearance via autophagy. Rab27b is localized to the autophagosome under stress conditions that induce autophagy (40). Rab27a/b double knockout mice show increased vesicle formation, including lysosomes and autophagosomes, in the lacrimal gland (41). Evidence for autophagic-lysosomal pathway impairment in PD is growing (42)(43)(44)(45)(46). The presence of Rab27b in the autophagiclysosomal process indicates the potential for Rab27b to alter not only ␣syn release but ␣syn clearance as well.
Rab27b has been linked to several neurodegenerative disorders. Elevated Rab27b expression in cholinergic basal forebrain neurons is associated with cognitive decline in mild cognitive impairment and Alzheimer's disease (47). In addition, multiple Rab27b polymorphisms have been associated with a higher risk for motor neuron disease in GWAS studies (48). Alterations in Rab27b expression are observed in a cellular model for X-linked dystonia parkinsonism syndrome and in human DLB brains (49,50).
Because of its function in regulating protein secretion and autophagy, we hypothesized that Rab27b regulates cell-to-cell transmission of ␣syn by regulation of ␣syn release and clearance. Here, we examine the effect of Rab27b on ␣syn toxicity in an in vitro paracrine ␣syn model and evaluate the impact of Rab27b on ␣syn release and clearance via autophagy. We found that Rab27b reduces ␣syn toxicity by promoting autophagic flux.

Rab27b reduces ␣syn-induced paracrine toxicity
We have developed a paracrine ␣syn model using a doxycycline (doxy)-inducible neuroblastoma cell line, termed isyn, to evaluate the toxicity associated with neuronally released ␣syn (Fig. 1a) (51). We have previously shown that induction of ␣syn expression with doxy in isyn cells leads to the detection of ␣syn in the CM in a dose-dependent manner and that fractionation of the CM into exosomal and nonexosomal fractions by sequential, high-ultracentrifugation techniques shows that ␣syn is released into both exosomal and nonexosomal fractions (51). Additionally, we have previously established that released ␣syn is toxic to separately cultured neurons: transfer of ␣synenriched CM from induced isyn cells promotes cell death of separately cultured differentiated SH-SY5Y neuroblastoma cells or primary mouse neurons (51). Toxicity from ␣syn-en- m. e, quantification of cell death in differentiated SH-SY5Y cells treated with CM from isyn cells transduced with EV, nt-shRNA, or Rab27b shRNA for 48 h. n ϭ 3 independent rounds with 1-2 replicates/round. One-way ANOVA: F (3,8) ϭ 44.90, p Յ 0.0001. Tukey's multiple-comparison test: ****, p Յ 0.0001. f, immunoprecipitation of ␣syn from CM by ␣syn-directed mAb reduces the toxicity of ␣syn-enriched CM from isyn/Rab27b KD cells in a dose-dependent manner. Shown is quantification of cell death in differentiated SH-SH5Y cells treated with CM from induced isyn/Rab27b KD cells in which ␣syn was immunodepleted. Western blotting of CM after immunoprecipitation confirms reduction of ␣syn in the CM. n ϭ 3 independent rounds with 1 replicate/round. One-way ANOVA: F (5,20) ϭ 39.10, p Յ 0.0001. Tukey's multiple comparison test: *, p Յ 0.05; **, p Յ 0.01; ***, p Յ 0.001; ****, p Յ 0.0001; ns, nonsignificant. g, Rab27b KD does not induce cell death in isyn cells. Shown is quantification of cell death in isyn transduced with nt-shRNA or Rab27b shRNA with and without doxy induction. The number of ethidium D-positive cells is normalized to total cell count, as determined by Hoechst 33342 staining. n ϭ 3 independent rounds with 1 replicate/round. One-way ANOVA: F (3,8)  Rab27b regulates ␣-synuclein toxicity riched CM depends on ␣syn, as immunodepletion of ␣syn from the CM eliminated toxicity in a dose-dependent manner (51). An initial PCR screen for detectable expression of potential exosome-related proteins in isyn cells pointed to Rab27b and Rab35 expression in isyn cells. Upon ␣syn induction with doxy (10 g/ml), Rab27b expression increased by nearly 2-fold, as determined by immunocytochemistry (Fig. 1b). We confirmed the increase in Rab27b by Western blotting (Fig. S1). No change was noted in Rab35 expression (Fig. S2a).
Given the increase in Rab27b expression upon ␣syn induction in isyn cells, we tested whether Rab27b knockdown (KD) could affect the toxicity of released ␣syn. isyn cells were transduced with Rab27b targeted shRNA lentivirus selectable by puromycin. Rab27b KD in isyn cells was confirmed by Western blotting (Fig. 1c). As controls for Rab27b KD, isyn cells were transduced with either plko.1 empty vector (EV) lentivirus or with nontarget shRNA (nt-shRNA) lentivirus. Toxicity of ␣syn-enriched CM was increased with Rab27b KD upon doxy induction in isyn cells compared with both isyn controls. Separately cultured differentiated SH-SY5Y showed ϳ10% cell death at 24 h when treated with CM from induced control isyn cells, but toxicity was doubled when treated with CM from induced isyn cells with Rab27b KD (Fig. 1, d and e). Increased toxicity from ␣syn-enriched medium from Rab27b KD/isyn cells depended on ␣syn, as immunodepletion of ␣syn from the CM eliminated toxicity in a dose-dependent manner (Fig. 1f). Rab27b KD did not impact cell death at baseline in isyn cells (Fig. 1g).

Rab27b regulates ␣syn release
Rab27b regulates protein secretion through both exosomal and nonexosomal pathways (29 -35). As Rab27b KD increased the toxicity of ␣syn released by isyn cells, we next examined whether Rab27b regulated the amount of ␣syn released in this paracrine model system. Surprisingly, upon doxy induction, we observed a nearly 60% decrease in the total amount of ␣syn released into the CM in isyn cells with Rab27b KD compared with control isyn cells transduced with nt-shRNA ( Fig. 2). Differences in release were not due to cell death, as cell death was limited to ϳ2-4% in induced isyn cells with and without Rab27b KD (Fig. 1g). These data indicate that the increase in ␣syn toxicity with Rab27b KD was not due to a total increase in ␣syn release.
Previous research has shown that ␣syn is released through exosomal and nonexosomal pathways, and certain studies have suggested that exosomally released ␣syn may have increased toxic potential (5,10,11,52). We fractionated the CM into exosomal and nonexosomal fractions by sequential, high-ultracentrifugation techniques (53) to test whether Rab27b KD possibly preferentially inhibited release through nonexosomal pathways. The vast majority of released ␣syn in our model is associated with the nonexosomal fraction from isyn cells (51). When Rab27b was knocked down in isyn cells, the amount of ␣syn released in the nonexosomal fraction was decreased by 44% compared with nt-shRNA control (Fig. 2e). The amount of ␣syn released in the exosomal fraction was also decreased relative to control isyn cells (Fig. 2f). NanoSight analysis confirmed nanometer-sized vesicle sizes consistent with exosomes released into the CM (51) (Fig. 2b). Thus, the total change in ␣syn release induced by Rab27b KD occurred through nonexosomal and exosomal pathways.

Rab27b promotes autophagic flux and ␣syn clearance
Our previous work in the paracrine ␣syn model revealed that the type of ␣syn species released was the critical factor that mediates toxicity, not the total amount of ␣syn released (51). This finding suggests that oligomeric, toxic ␣syn species are potentially available for release. To biochemically characterize ␣syn released into the CM, we used size-exclusion chromatography (SEC). We and others have previously shown that much of the released ␣syn is found in highermolecular-weight fractions representing molecular sizes greater than 14 kDa, the expected monomeric ␣syn size by SEC (5,51,54). We fractionated CM from induced nt-shRNA/isyn cells and induced isyn/Rab27b KD cells by SEC and found that the amount of released ␣syn released into higher-molecular-weight fractions was significantly increased by Rab27b KD (Fig. 3, a and b). ELISA of total CM prior to SEC fractionation confirmed that total ␣syn levels was overall lower in the CM from isyn cells with Rab27b KD compared with that from induced nt-shRNA isyn cells despite the increase in higher molecular weight ␣syn (Fig. S3).
Given the increase in high-molecular weight ␣syn species released in the presence of Rab27b KD, we hypothesized that Rab27b promotes the clearance of intracellular ␣syn. We next examined whether insoluble ␣syn levels were altered in induced isyn cells upon Rab27b KD. Whereas ␣syn levels in the Triton X-100 soluble fraction did not statistically differ, we observed more ␣syn in the Triton X-100 -insoluble fraction in isyn cells with Rab27b KD (Fig. 3c).
Misfolded proteins are typically cleared through the autophagic pathway or through the proteasome. ␣syn, particularly oligomeric species, has been previously shown to be degraded through autophagy (46,55). We hypothesized that Rab27b promotes ␣syn clearance via the autophagic-lysosomal pathway. We first examined levels of proteins involved in the endosomal-lysosomal system in doxy-induced isyn with and without Rab27b KD cells under serum starvation. Rab7, a late endosome marker, and p62, an autophagy marker, were increased in induced isyn cells with Rab27b KD, whereas the early endosome marker Rab5 remained unchanged (Fig. 4). Similarly, LC3-positive puncta were increased in induced isyn cells with Rab27b KD under serum starvation compared with induced control isyn cells (Fig. 5a).
To further test the impact of Rab27b on autophagic flux, control isyn and isyn/Rab27b KD cells were induced with doxy for 96 h in serum-free medium followed by treatment with 40 M chloroquine for 3 h. Chloroquine inhibits autophagosome/lysosome fusion, leading to an accumulation of LC3II in normal cells under autophagy-inducing conditions. Levels of LC3 II were increased in control isyn cells with chloroquine treatment, demonstrating an increase in autophagic flux (Fig. 5b). However, chloroquine failed to induce an increase in LC3 II in the presence of Rab27b KD in isyn cells (Fig. 5b). This finding points to a defect in autophagic flux with Rab27b KD. Partial colocalization of Rab27b with LC3 Rab27b regulates ␣-synuclein toxicity Figure 2. Rab27b knockdown reduces ␣syn release into the CM. a, representative Western blots of total ␣syn levels in CM and lysates from uninduced and induced isyn cell lines transduced with EV, nt-shRNA (NT), or Rab27b shRNA after doxy (10 g/ml) induction for 96 h. Equal protein levels were loaded for each CM sample. b, NanoSight analysis shows that exosomal fraction from CM of control nt-shRNA-transduced isyn cells contains particles averaging 106 nm, consistent with the size of exosomes, after 96 h of induction with doxy. c, quantification of ␣syn in the intracellular lysates from isyn cell lines transduced with EV, nt-shRNA, or Rab27b shRNA with and without 96-h induction by Western blotting. ␣syn in lysates was normalized to tubulin. n ϭ 3 independent rounds. One-way ANOVA: F (5,12)

Rab27b regulates ␣-synuclein toxicity
and with LAMP1 in isyn cells demonstrates that Rab27b can interact with autophagosomes, lysosomes, and/or autolysosomes (Fig. 5, c and d). Together, these data suggest that Rab27b normally promotes autophagic ␣syn clearance and that impaired autophagic clearance of ␣syn by Rab27b KD promotes ␣syn toxicity.

Rab27b levels are increased in human PD and DLB
We evaluated Rab27b protein expression in the postmortem brain lysates in the medial temporal gyrus from age-matched controls and subjects with clinically and pathologically diagnosed PD or DLB. Rab27b levels were increased in the postmortem, Triton X-100 -soluble lysates of PD patients 2.1-fold compared with controls (Fig. 6a). Rab35 levels were not altered in PD brains compared with controls (Fig. 2b). Rab27b levels in the postmortem lysates of DLB patients were also increased by 20% compared with controls (Fig. 6b).

Discussion
Our data demonstrate that Rab27b regulates the release, clearance, and toxicity of ␣syn in a cellular paracrine model. We observed that Rab27b KD in isyn cells increased ␣syn paracrine toxicity. Rab27b KD induced a paradoxical decrease in ␣syn release, but the lower levels of released ␣syn were of highermolecular-weight species. Rab27b KD also increased intracellular insoluble ␣syn levels. We conclude that Rab27b KD leads to an increase in ␣syn paracrine toxicity due to a reduction of clearance of misfolded ␣syn through autophagy. Consistent with this, Rab27b KD led to increased LC3-positive autophagosome accumulation and p62 levels and inhibited autophagic flux. Together, these data suggest that Rab27b plays an integral role in the release, clearance, and toxicity of ␣syn.
We have previously published on the advantages of our paracrine in vitro model that allows us to examine distinct parts of the various processes required for the prion-like spread of ␣syn Rab27b regulates ␣-synuclein toxicity (51). The studies detailed above indicate the potential cellular mechanisms by which Rab27b regulates ␣syn spread and toxicity in this model. Our observations point to Rab27b as a regulator of ␣syn propagation through multiple cellular mechanisms. Although Rab27b KD inhibited ␣syn release, it also decreased autophagic flux, and released ␣syn was more likely to promote toxicity. Despite the reduction in total amount of released ␣syn, a probable shift to species capable of templating the misfolding of endogenous ␣syn contributed to the increase in toxicity in cells treated with ␣syn-enriched CM from isyn/ Rab27b KD cells. Our previous studies have shown that the ␣syn released into the CM is primarily oligomeric and promotes seeding (51). Given that insoluble ␣syn was enhanced by Rab27b KD, our data suggest that the disruption of autophagy led to an increase in oligomeric, toxic ␣syn release.
Together, these results indicate that Rab27b may function as an endogenous regulator of ␣syn clearance via autophagy and release. Under normal conditions, we propose that Rab27b clears misfolded ␣syn to prevent the intracellular buildup of toxic oligomers that can promote seeding by 1) promoting autophagic clearance and 2) promoting ␣syn release (Fig. 7a). Disruption of Rab27b function allows for the accumulation of intracellular misfolded ␣syn (Fig. 7b). Although total ␣syn secretion is decreased by Rab27b depletion, any ␣syn that is released has a higher seeding capacity and can then be taken up by neighboring neurons to induce pathologic seeding of endogenous ␣syn (Fig. 7b). Rab27b depletion led to a decrease in ␣syn release in both exosomal and nonexosomal fractions, indicating that multiple release mechanisms are regulated by Rab27b and may contribute to disease progression.
Rab27b levels were increased in the postmortem brain lysates of PD patients compared with age-matched healthy controls.
We also found that Rab27b protein levels were increased in the brain lysates of DLB patients as well, in accordance with previously published transcriptome data (50). We propose that these increases in expression may be compensatory in nature. As intracellular misfolded protein accumulates, neurons may upregulate Rab27b to increase aggregated protein clearance through the autophagic pathway. Because Rab27b also promotes ␣syn release, any increase in Rab27b in disease could theoretically promote ␣syn transmission from cell to cell. However, as we have previously published, an increase in total amount of ␣syn released into the CM does not necessarily correlate to increased paracrine toxicity but is instead dependent on the conformation of released ␣syn (51). Indeed, our data show that Rab27b KD actually increased the toxicity of released ␣syn despite lower total ␣syn amounts in the CM; increased toxicity was likely due to the release of higher-molecularweight species secondary to disrupted autophagic clearance.
The molecular mechanisms by which Rab27b regulates autophagic clearance and protein secretion are unclear at this time. Rab27b has been shown to promote distal transport and docking of secretory vesicles, including lysosomes, with the plasma membrane (28,29,31,33,36,37,41,56,58). Rab27b could potentially promote lysosomal fusion with the plasma membrane to promote ␣syn secretion. Additionally, Rab27b could promote autophagic-lysosomal clearance of ␣syn by promoting distal transport of lysosomes and fusion with autophagosomes. Consistent with this, the autophagic flux assay with the lysosomal inhibitor CQ (Fig. 5b) suggests that Rab27b does act at later stages in the autophagic-lysosomal pathway. Our data showing that Rab27b partially colocalizes with LAMP1 and LC3 point to its potential localization in autophagosomes, lysosomes, and/or fused autolysosomes (Fig. 5, c and d).
Rab effectors associated with Rab27b that are highly expressed in the brain include Slp5, Slp2a, rabphilin, Slac2a, and myrip (30), and we predict that different effectors are involved in the differential regulation of autophagic-lysosomal clearance versus secretion by Rab27b. Of these effectors, Slp5 (Sytl5) has been previously identified through shRNA screen by Goncalves et al. to regulate ␣syn release (18). Other protein partners that may interact with Rab27b include motor proteins and SNARE proteins to promote vesicular transport and fusion. Motor proteins and adaptors are required partners for Rab proteins to aid transport (59,60), and Rab27 isoforms have been shown to interact with kinesins to promote anterograde transport of secretory lysosomes and TrkBϩ vesicles (38,61). Once Rab27b has potentially brought lysosomes into position to fuse with the plasma membrane or autophagosomes, fusion would likely require tethering and SNARE proteins (59,60).
Future directions will focus on these endolysosomal players that may interact with Rab27b, with a focus on the critical Rab27b effectors that regulate Rab27b's differential effects on autophagy and secretion. Testing the role of Rab27b in rodent or iPSC-based models of synucleinopathies will also be important in understanding the role of Rab27b in human disease. Whereas in vitro models are useful tools for testing basic cellular mechanisms, validation of these findings in more complex models is critical, given the limited biological complexity of cellular models.

Rab27b regulates ␣-synuclein toxicity
In conclusion, Rab27b regulates ␣syn toxicity in our paracrine model and is up-regulated in PD and DLB. Targeting Rab27b function could be a target for therapeutic intervention in these disorders.

Human brain samples
Human brain tissue was obtained from deceased persons, and the use of the human specimens was reviewed by the institutional review board at the University of Alabama at Birmingham and determined to be not human subjects research and not subject to Food and Drug Administration regulation. Sample identification was blinded and not available to the investigators. This work abides by the Declaration of Helsinki principles.

Preparation of CM
CM was prepared as described previously (51). After serial centrifugations to remove cellular debris, CM was concentrated using a 3-kDa Amicon Ultra-4 centrifugal filter at 4000 ϫ g for 2 h, followed by dialysis. Protein concentrations of CM samples were assessed by BCA assay (Thermo Fisher Scientific), and equal protein amounts were loaded for each CM sample for Western blot analysis.
For toxicity experiments, isyn cells were induced with doxy in Eagle's MEM/F12K with 10% FBS for 1 week and then switched to serum-free Eagle's MEM/F12K for 48 h. Collected CM underwent centrifugation at 800 ϫ g for 5 min and then at 2000 ϫ g for 10 min and then at 10,000 ϫ g for 30 min prior to transfer to differentiated SH-SY5Y cells.

Ethidium D cell death assay
Cells were rinsed in PBS and then incubated in 1 M ethidium D and 2 g/ml Hoechst 33342 in culture medium for 20 min at 37°C. Ten high-power (ϫ20) fields per well were randomly selected for quantification, and the number of ethidium D-positive cells and the total number of cells stained  (2), where the ␣syn can be taken up by other neurons (3). b, upon Rab27b knockdown, intracellular ␣syn aggregation increases by impaired autophagic clearance of ␣syn (4). Whereas Rab27b KD decreases total ␣syn release, the released ␣syn has higher seeding potential (5), resulting in higher toxicity in neurons that take up the released ␣syn (6).

Rab27b regulates ␣-synuclein toxicity
by Hoechst 33342 were counted per high power field with the rater blind to experimental conditions.

Autophagic flux assay
isyn cells infected with nontargeted shRNA or Rab27b shRNA were induced with doxy at 10 g/ml for 96 h in serumfree Eagle's MEM/F12K medium. Cells were then treated with vehicle or 40 M chloroquine for 3 h at 37°C prior to collection of cell lysates.

Western blotting
Western blot analysis was performed as described previously (51). Equal protein amounts were loaded per well for the CM samples and for cell lysate samples. Primary antibodies used are listed in Table 1. Blots were developed with the enhanced chemiluminescence method (GE Healthcare). Images were scanned using the Bio-Rad Chemidoc Imaging System and analyzed using Image Lab Bio-Rad software for densitometric analysis of bands.
Fresh-frozen tissue from temporal cortices of age and gender-matched control, PD, and DLB brains were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program. Samples were prepared as described previously (57). Rab27b protein levels were normalized to total protein levels determined by SYPRO Ruby protein gel stain (Invitrogen).

Exosome fractionation
For exosome preparation, we followed the protocol described previously (51,53). Briefly, cells were incubated to serum-free Eagle's MEM/F12K for 96 h. CM underwent serial centrifugations at 800 ϫ g for 5 min, 2000 ϫ g for 10 min, and 10,000 ϫ g for 30 min to remove cellular debris at 4°C. Debrisfree medium was then spun at 100,000 ϫ g for 2 h at 4°C. The supernatant was saved as the nonexosomal fraction. The pellet was resuspended in PBS and then spun again at 100,000 ϫ g for 80 min at 4°C. The pellet was resuspended in PBS with protease inhibitors.

NanoSight
The size of exosomes was determined on a NanoSight NS300 (Malvern Instruments, Westboro, MA), as described previously (51). Following isolation by ultracentrifugation, exosome pellets were resuspended in 60 l of cold PBS with repeated pipetting and vortexed for 10 s. Then 15 l of the exosome suspension was diluted to a total volume of 1 ml of PBS and analyzed on a NanoSight NS300 infused with a syringe pump set at 25 (arbitrary units). Data were collected for each sample in 10 repeats of a 60-s video and analyzed using NanoSight NTA 3.0 software.

Size-exclusion chromatography
SEC was performed as described previously (51). 20 l (80 g) of CM diluted in PBS was loaded onto an NGC FPLC (Bio-Rad), injected on a Yarra 3-m SEC-2000 column (300 ϫ 7.8 mm; Phenomenex), and run at 0.7 ml/min in 1ϫ PBS, pH 6.8. 250-l fractions were collected from elution volume 4 -12.5 ml. This corresponds to the end of the void volume, as determined by a blue dextran standard, and the buffer front, as determined by imidazole elution. ␣syn in 50 l of each fraction was measured using an ELISA for ␣syn.

Immunocytochemistry
isyn cells were fixed in 4% paraformaldehyde for 15 min. After washing in PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then blocked with 5% NGS in PBS for 20 min. Cells were incubated overnight with primary antibody (Rab27b, Rab5, Rab7, LC3II, p62, Golgin-97, LAMP1) Rab27b regulates ␣-synuclein toxicity in 1.5% NGS. Primary antibodies used are described in Table 1. After washing, cells were incubated with goat anti-rabbit or anti-mouse secondary antibody in 1.5% NGS for 2 h. isyn cells were imaged using an Olympus BX51 epifluorescence microscope. Ten high-power (ϫ20) fields per well were randomly selected for quantification, and the immunoreactivity was quantitated using ImageJ with the rater blind to experimental conditions. For LC3II puncta counts, slides were imaged at ϫ63 by confocal microscopy (Leica TCS-SP5 laser-scanning confocal microscope) and quantitated using an ImageJ cell counter. For Rab27b colocalization, Z-stack images of neurons were taken at ϫ63 by confocal microscopy (Nikon Eclipse Ti2 scanning confocal microscope).

Experimental design and statistical analysis
GraphPad Prism 8 (La Jolla, CA) was used for statistical analysis of experiments. Data were analyzed by either Student's t test or one-way or two-way ANOVA, followed by post hoc pairwise comparisons using Sidak's or Tukey's multiple-comparison tests. Statistical significance was set at p Յ 0.05. ANOVArelated statistics (F statistic, p values) and post hoc test results are found in the figure legends. For t tests, the t statistic and p values are noted in the figure legends.

Data availability
All data are contained in the article.