Rab27b Promotes Lysosomal Function and Alpha-Synuclein Clearance in Neurons
- Kasandra Scholz*,
- Rudradip Pattanayak*,
- Roschongporn Ekkatine,
- F. Sanders Pair,
- Amber Nobles,
- William J. Stone, and
- Talene A. Yacoubian
- Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294
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Author contributions: K.S., R.P., and T.A.Y. designed research; K.S., R.P., R.E., A.N., and W.J.S. performed research; K.S., R.P., F.S.P., W.J.S., and T.A.Y. analyzed data; K.S. and R.P. wrote the paper.
Abstract
Alpha-synuclein (αsyn) is the key pathogenic protein implicated in synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In these diseases, αsyn is thought to spread between cells where it accumulates and induces pathology; however, mechanisms that drive its propagation or aggregation are poorly understood. We have previously reported that the small GTPase Rab27b is elevated in human PD and DLB and that it can mediate the autophagic clearance and toxicity of αsyn in a paracrine αsyn cell culture model. Here, we expanded our previous work and characterized the role of Rab27b in neuronal lysosomal processing and αsyn clearance. We found that Rab27b KD in this αsyn-inducible neuronal model resulted in lysosomal dysfunction and increased αsyn levels in lysosomes. Similar lysosomal proteolytic defects and enzymatic dysfunction were observed in both primary neuronal cultures and brain lysates from male and female Rab27b knock-out (KO) mice. αSyn aggregation was exacerbated in Rab27b KO neurons upon treatment with αsyn preformed fibrils. We found no changes in lysosomal counts or lysosomal pH in either model, but we did identify changes in acidic vesicle trafficking and in lysosomal enzyme maturation and localization, which may drive lysosomal dysfunction and promote αsyn aggregation. Rab27b OE enhanced lysosomal activity and reduced insoluble αsyn accumulation. Finally we found elevated Rab27b levels in human postmortem incidental Lewy body disease subjects relative to healthy controls. These data suggest the role of Rab27b in neuronal lysosomal activity and identify it as a potential therapeutic target in synucleinopathies.
Significance Statement
Alpha-synuclein (αsyn) aggregation in Parkinson's disease is associated with autophagic–lysosomal dysfunction, yet the molecular mechanisms underlying αsyn clearance are not well understood. We identified the small GTPase Rab27b as a novel regulator of the lysosomal clearance of αsyn. Using several αsyn models, we found that Rab27b knockdown or knock-out impairs lysosomal function, increases αsyn lysosomal accumulation, and increases αsyn aggregation. Conversely, Rab27b overexpression promotes lysosomal function and reduces αsyn aggregation. We also identified defects in lysosomal enzyme maturation and localization and acidic vesicle trafficking upon Rab27b loss, which may drive lysosomal dysfunction. These findings suggest that targeting Rab27b could boost lysosomal clearance of αsyn in synucleinopathies.
Introduction
As the global population ages, synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are expected to increase dramatically in prevalence, exacerbating the existing social and economic burdens associated with patient care (Dorsey et al., 2007; Kaltenboeck et al., 2012; GBD 2016 Parkinson's Disease Collaborators 2018). These diseases are pathologically characterized by intracellular proteinaceous inclusions comprised of alpha-synuclein (αsyn), which misfolds, spreads between cells, aggregates, and induces pathology (Spillantini et al., 1997; Chang et al., 2017). It is thought to spread in a prion-like manner where toxic, oligomeric species contact healthy monomeric αsyn and template its misfolding (Danzer et al., 2009; Leak et al., 2019), but the mechanisms governing this spread and accumulation remain poorly understood.
Various biological processes likely contribute to the spread and aggregation of αsyn, including autophagic and endolysosomal pathways. These pathways are active areas of interest in PD; both monomeric and misfolded αsyn can be degraded by autophagic processes (Vogiatzi et al., 2008; Martinez-Vicente and Vila, 2013), PD pathology is associated with autophagic–lysosomal dysfunction (Lynch-Day et al., 2012; Chang et al., 2017), and upregulating autophagy may have therapeutic potential (Moors et al., 2017; Lu et al., 2020). In disease states, dysregulated autophagy may impair αsyn clearance and thereby facilitate aggregation and cell death (Zhang et al., 2015; Hou et al., 2020; Fellner et al., 2021). Furthermore, αsyn itself may damage autophagic capacity, resulting in a positive feedback loop that can exacerbate pathology (Xilouri et al., 2016; Lei et al., 2019). Within these pathways, lysosomes are key structures of interest, and lysosomal impairment may be a major contributor to PD pathogenesis (Dehay et al., 2013; Wallings et al., 2019; Navarro-Romero et al., 2020). Several genetic lysosomal defects result in or increase the risk of PD, including mutations in the lysosomal ATPase ATP13A2 and the lysosomal enzyme GBA1 (Dehay et al., 2012; Do et al., 2019). Autophagic–lysosomal pathways, which are regulated in part by members of the Rab family of GTPases, may therefore play a critical role in disease progression.
Rab GTPases are a family of over 60 proteins that regulate a variety of cellular processes including trafficking, endocytic, secretory, and autophagic–lysosomal pathways (Pereira-Leal and Seabra, 2001; Stenmark, 2009; Homma et al., 2021). Several members of this family have been implicated in neurodegeneration and PD, both as direct modulators of αsyn and targets of the PD-associated kinase LRRK2 (Gonçalves et al., 2016; Kiral et al., 2018; Bonet-Ponce and Cookson, 2019; Lara Ordóñez et al., 2021). Rab27b is a member of this family that has been implicated in multiple cellular processes including exosome secretion and synaptic transmission (Mahoney et al., 2006; Ostrowski et al., 2010). We have previously identified Rab27b as a regulator of the release, toxicity, and clearance of αsyn in an in vitro inducible human αsyn model (Underwood et al., 2020). Additionally, we found an increase in Rab27b levels in the temporal cortex of human PD and DLB patients relative to age-matched controls (Underwood et al., 2020). Here we describe the new role of Rab27b in the lysosomal function of neurons and in the lysosomal clearance of αsyn. We evaluated Rab27b's role in lysosomal function and αsyn pathology using knockdown (KD), knock-out (KO), and overexpression (OE) models in vitro and in vivo. We found that KD or KO of Rab27b impairs lysosomal function and exacerbates αsyn pathology, while OE conversely enhances lysosomal functioning and ameliorates pathology, establishing Rab27b as a regulator of autophagic–lysosomal processing in the context of synucleinopathies.
Materials and Methods
Cell lines and mouse models
Cell lines
We previously developed a doxycycline (doxy)-inducible SK-N-BE(2)-M17 cell line (isyn) that overexpresses and secretes αsyn into the culture media upon treatment with doxy, as previously described (Wang et al., 2018). To reduce Rab27b expression, we transduced isyn cells with a lentiviral Rab27b-targeted shRNA (5′-CCCAAATTCATCACTACAGTA-3′) to knock down Rab27b or with a lentiviral nontarget (nt) shRNA (Sigma-Aldrich SHC016) as a control as previously described (Underwood et al., 2020).
To test the effect of Rab27b OE, we transduced isyn cells with a lentiviral Rab27b-GFP construct (Genecopoeia EX-Q0376-Lv103) to overexpress Rab27b or a lentiviral GFP-expressing construct (GeneCopoeia EX-NEG-Lv103) as a control. Cells were selected for stable transfection with puromycin (1 µg/ml).
All isyn-based cell lines were treated with 500 µg/ml G418 sulfate to maintain selection for inducible αsyn constructs and with 1 µg/ml puromycin to maintain selection for nt-shRNA, Rab27b KD shRNA, GFP, and Rab27b OE constructs. Cells were maintained in a 1:1 ratio of F-12K and EMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. To induce αsyn OE, we treated cells with 10 µg/ml doxy hyclate (Slone et al., 2015).
Rab27b KO mice
Mice were used and maintained in accordance with University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee protocols and the guidelines of the National Institute of Health. Rab27a and Rab27b double KO (DKO) mice acquired from Miguel Seabra (Tolmachova et al., 2007) were crossed with C57BL/6-J mice from Jackson Laboratory (000664) to generate a single KO mouse for Rab27b. Rab27 DKO mice were crossed with wild-type (WT) mice to generate Rab27a+/− Rab27b+/− progeny, which were then crossed to each other to generate Rab27a+/+ Rab27b+/− mice. For maintenance breeding, Rab27b+/− heterozygous mice were bred together to generate litters with both WT control (Rab27b+/+) and Rab27b KO (Rab27b−/−) pups for experimental use. Both male and female animals were used. Mice were genotyped for Rab27b and αsyn Exon 4 as previously described (Pattanayak et al., 2022).
Rab27b KO a53T transgenic mice
Rab27b KO (Rab27b−/−) mice were crossed with A53T hemizygous (A53T+/−) mice (Lee et al., 2002) acquired from Jackson Laboratory (006823) to generate progeny that were Rab27b heterozygous (Rab27b+/−) and either A53T hemizygous (A53T+/−) or A53T negative (A53T−/−). These progeny were then crossed to generate the following gene combinations: Rab27b+/+ A53T−/−, Rab27b+/+ A53T+/−, Rab27b−/− A53T−/−, and Rab27b−/− A53T+/−. These animals were used to set up final crosses in two parallel lines: a Rab27b WT line (Rab27b+/+ A53T+/−×Rab27b+/+ A53T−/−) and a Rab27b KO line (Rab27b−/− A53T+/−×Rab27b−/− A53T−/−). Pups from these parallel cages were cultured together when born on the same day for experiments involving all four final genotypes: Rab27+/+ A53T−/− (WT mice); Rab27b+/+ A53T+/− (A53T+ mice); Rab27b−/− A53T−/− (Rab27b KO mice); and Rab27b−/− A53T+/− (Rab27b KO/A53T+ mice). Both male and female animals were used.
Primary neuronal cultures
Primary neurons were harvested from postnatal day (P)0 pups as described previously (Wang et al., 2018). Briefly, P0 pups were anesthetized on ice before brain collection. Hippocampi were bilaterally isolated and digested with papain for 30 min at 37°C. Hippocampi were mechanically dissociated and cells were plated in Neurobasal A media supplemented with B27, GlutaMAX, FBS, and penicillin–streptomycin. At 24 h, cells were treated with 1 µm AraC in media without FBS to inhibit glial proliferation.
Immunocytochemistry
Isyn cells were plated on glass coverslips in 24-well plates at 10,000 cells per well and treated with or without 10 µg/ml doxy for 96 h before being harvested by fixation in a 4% paraformaldehyde (PFA)/4% sucrose solution. Following washing, cells were permeabilized in 0.1% Triton X-100 in 3% BSA for 20 min followed by a 1 h block in 3% BSA. Cells were incubated in primary antibodies (Rab27b, LAMP-1, LC3B, p62, EEA1, Rab7, αsyn, Ctsd) in 3% BSA overnight at 4°C. Primary antibodies are described in Table 1. Cells were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies in 3% BSA for 1 h before being washed and mounted on slides with ProLong Diamond DAPI mounting media for imaging on a confocal microscope at 63× (Nikon Eclipse Ti2 scanning confocal microscope). Pearson's correlation coefficients were quantified using Nikon NIS-Elements. Primary neurons were plated in Xona microfluidic dual-chamber devices (Table 2) and allowed to grow until day in vitro (DIV) 7, at which point axons had grown through the chambers and could be isolated. They were then stained and imaged as described above.
Primary antibodies used
Other reagents used
Proximity ligation assay (PLA)
Isyn cells were plated on glass coverslips in 24-well plates at 10,000 cells per well and treated with 10 µg/ml doxy for 96 h before being harvested by fixation in 4% PFA for 20 min at room temperature. PLA was executed according to the manufacturer's protocols. Briefly, cells were incubated in primary antibody at 4°C overnight (Table 1) and then washed and treated with Duolink PLA PLUS and MINUS probes (Table 2) before amplification and coverslipping. Cells were imaged on a confocal microscope at 63× as described above.
Lysosomal fractionation: isyn/Rab27b KD cells
Isyn/Rab27b KD or control cells were treated with 10 µg/ml doxy for 96 h before being harvested for lysosomal fractionation using a Percoll density gradient as previously described (Carosi et al., 2019). Briefly, cells were manually lysed and added to a sucrose-Percoll gradient before being spun at 36,000 × g for 30 min (Beckman Coulter Optima XE-90 Ultracentrifuge; SW 31 Ti rotor). Lysosomal fractions were manually collected and used for Western blot analysis. Blots were probed for LAMP-1, αsyn, GAPDH, and histone H1 (Table 1).
Lysosomal fractionation: isyn/Rab27b OE cells
Isyn/Rab27b OE or isyn/GFP control cells were treated with 10 µg/ml doxy for 96 h before being harvested for lysosomal fractionation using a Lysosome Enrichment Kit (Table 2). Briefly, cells were lysed and added to an OptiPrep density gradient before being spun at 145,000 × g for 2 h (Beckman Coulter Optima XE-90 Ultracentrifuge; SW 31 Ti rotor). Lysosomal fractions were manually collected, washed, and then further lysed to free lysosomal proteins before Western blot analysis. Blots were probed for LAMP-1, αsyn, GAPDH, and histone H1 (Table 1).
Lysosomal enzyme assays
Cathepsin D activity
Isyn-based cells were treated with 10 µg/ml doxy for 96 h before being lysed in RIPA buffer to extract proteins. A 5 µg of protein was incubated with either 40 µM cathepsin D (Ctsd) Substrate or 40 µM Ctsd Substrate plus 30 µM Substrate Inhibitor Pepstatin A diluted in 50 mM sodium acetate, pH 4.0, for 60 min at 37°C in 96-well plates. Plates were read on a SpectraMax iD3 (Molecular Devices) plate reader (EX, 360 nm; EM, 460 nm), and substrate wells were normalized to substrate and inhibitor wells. Similarly, brain lysates from WT and Rab27b KO mice were incubated with Ctsd Substrate with and without pepstatin A to measure Ctsd activity in vivo.
Glucocerebrosidase activity
Isyn-based cells were treated and lysed as described above. Glucocerebrosidase (GCase) activity was measured as previously described (Arrant et al., 2019). Briefly, 5 µl of each sample was added to a 96-well plate and incubated with 45 µl of GCase assay buffer (1.11% BSA, 0.28% Triton X-100, 0.28% taurocholic acid, 1.11 mM EDTA) containing either 1.11 mM 4-methylumbelliferone β-d-glucopyranoside (4-MUGluc) or 1.11 mM 4-MUGluc with 0.2 mM conduritol B epoxide (CBE). Plates were incubated at 37°C for 1 h, after which 50 µl of stop solution (0.4 M glycine), pH 10.8, was added to each well. Fluorescence intensity was recorded on a SpectraMax iD3 (Molecular Devices) plate reader (EX, 355 nm; EM, 460 nm), and substrate wells were normalized to CBE inhibitor wells and then to total protein levels. Similarly, brain lysates from WT and Rab27b KO mice were incubated with 4-MUGluc Substrate with and without CBE to measure GCase activity in vivo.
GCase endoglycosidase H (EndoH) assay
GCase maturity was assessed using an EndoH assay as described (Cuddy and Mazzulli, 2021). Briefly, isyn/Rab27b KD and control cells were induced with 10 µg/ml doxy for 96 h prior to lysis in lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 25 mM HEPES at pH 7.4, 1 mM EDTA, 1.5 mM MgCl2). A 50 µg of protein from each sample was then denatured and digested with EndoH (Table 2). Control samples were not EndoH treated. Samples were assessed via Western blot and probed with GCase antibody (Table 1).
Lysosomal acid lipase activity
Isyn-based cells were plated on 35 mm MatTek live imaging dishes (Table 2) and treated with 10 µg/ml doxy for 96 h. Cells were then treated with lysosomal acid lipase (LAL) substrate (Table 2) for 6 h before being imaged live on a Carl Zeiss Observer high-speed live cell imaging system microscope. For examination of LAL activity in primary hippocampal neurons, neurons were treated at DIV 8 with LAL substrate prior to imaging.
DQ-BSA assay
Isyn-based cells were plated on 35 mm MatTek live imaging dishes and treated with 10 µg/ml doxy for 96 h. Cells were then treated with DQ-BSA substrate and imaged after 6 h using either a Carl Zeiss Observer high-speed live cell imaging system or Nikon Eclipse Ti2 scanning confocal microscope. WT and Rab27b KO primary hippocampal neurons were treated at DIV 8 prior to treatment and imaging.
LysoTracker staining
Isyn-based cells were plated on 35 mm MatTek live imaging dishes and treated with 10 µg/ml doxy for 96 h. Cells were then treated with 200 nM LysoTracker Red (Table 2) and incubated for 1 h. Cells were then live-imaged at 63× using a Carl Zeiss Observer high-speed live cell imaging system.
Lysosomal pH assays
Isyn/Rab27b KD and control cells were plated on 35 mm MatTek live imaging dishes and treated with 10 µg/ml doxy for 96 h. Cells were then stained using a lysosomal pH kit from Dojindo Laboratories according to the manufacturer's protocols (Table 2). Cells were washed twice in serum-free media and then incubated in LysoPrime Green stain at 1:2,000 for 30 min at 37°C to mark total lysosomes. Cells were then washed twice and incubated in pHLys Red stain at 1:1,000 for 30 min at 37°C to mark acidified lysosomes. Cells were washed two more times, incubated with DAPI at 1:2,000 for 5 min at 37°C, washed two more times, and then live-imaged in fresh media at 63× on a confocal microscope (Nikon Eclipse Ti2 scanning confocal microscope). Control cells incubated with bafilomycin A (BafA) at 1:1,000 were treated with LysoPrime Green as described above and then treated with a solution combining pHLys Red and BafA to inhibit lysosomal acidification before undergoing washes and imaging as described.
Triton X-100 fractionation
Isyn cells were treated with 10 µg/ml doxy for 96 h before being harvested. Control cells did not receive doxy. Cells were first resuspended and sonicated in lysis buffer (50 mM Tris–HCL pH 7.4, 175 mM NaCl, and 5 mM EDTA pH 8.0) with 1% Triton X-100 and spun for 60 min at 15,000 × g to extract Triton X-100–soluble proteins. Pellets were then resuspended in lysis buffer containing 2% SDS and sonicated to extract Triton X-100–insoluble proteins. Mouse brains were homogenized and sonicated in lysis buffer with 1% Triton X-100, incubated on ice for 30 min, and spun for 60 min at 15,000 × g to extract Triton X-100–soluble proteins. Pellets were then resuspended in lysis buffer containing 2% SDS and sonicated to extract Triton X-100–insoluble proteins. Samples were assessed for αsyn and GAPDH by Western blot (Table 1).
Conditioned media αsyn release assay
Isyn/Rab27b OE and GFP control cells were plated in P100 dishes at 1.5 million cells per dish. Cells were treated with or without 10 µg/ml doxy for 96 h in serum-free media. Media were harvested and treated with 0.1 M PMSF before being concentrated using a 3 kDa cutoff Amicon filter tube (Table 2). Concentrated conditioned media were assessed via Western blot for αsyn, and total protein was assessed on a duplicate gel using BlueSafe gel stain (Table 2). Concurrent with media processing, cells were lysed in 0.5% NP-40 and assessed for αsyn and GAPDH via Western blot.
Dextran internalization assay
WT and Rab27b KO primary hippocampal neurons were plated on 35 mm MatTek dishes. At DIV 7, cells were placed on ice, washed twice in Neurobasal A–phenol red media, and treated with 50 mg/ml dextran (10,000 MW) (Table 2). Cells were left on ice for 30 min to allow dextran to settle and were then moved to a 37° incubator for 30 or 60 min to allow endocytosis to occur. As controls, WT cells were either incubated on ice with 50 mg/ml dextran for 90 min or preincubated with 6 µM cytochalasin D (CytD) for 60 min preceding treatment with 50 mg/ml dextran/6 µM CytD for 30 min on ice followed by 60 min at 37°. Plates were washed twice with Neurobasal A–phenol red, and extracellular signal was quenched with 1 mM trypan blue for 3 min before cells were live-imaged on a Nikon Eclipse Ti2 scanning confocal microscope.
Similarly, isyn/Rab27b KD and control cells were plated on 35 mm MatTek dishes at 10,000 cells per dish and induced with 10 µg/ml oxy for 96 h before undergoing dextran treatment as described above. Isyn CytD controls were treated at 3 µM.
Lysosomal trafficking assay
WT and Rab27b KO primary hippocampal neurons were cultured on 35 mm MatTek dishes. At DIV 10, cells were exposed to 200 nM LysoTracker Red for 1 h. Axons from pyramidal neurons were identified based on their growth cone morphology, lack of spines, and nontapering neurite structure. Axons were imaged using a Zeiss Z1 Cell Observer (63× oil, 1.4 NA) equipped with a Colibri2 cool LED five-channel fast wavelength switching system and a Hamamatsu Orca Flash high-speed camera to assess axonal transport. Time-lapse videos were acquired at 3 frames/sec × 2 min. Videos of the axons were recorded for >100 µm from the soma and from the axon terminal/synapses. Kymographs from time-lapse videos were generated using KymoAnalyzer (Neumann et al., 2017). This software provides parameters including the percentage of cargo in anterograde, retrograde, reversing, or stationary motion; the percentage of the time spent in each type of motion; the number and frequency of pauses; the number and frequency of switches in direction; as well as net and segmental velocities.
Lysosomal exocytosis assay
Isyn/Rab27b KD and control cells were plated on glass coverslips at 10,000 cells per coverslip and induced with 10 µg/ml doxy for 96 h. Cells were stained as previously described (Samie et al., 2013; Tsunemi et al., 2019). Briefly, cells were treated with 200 nm BafA1 for 2 h to stimulate lysosomal exocytosis and then incubated in LAMP-1 primary antibody for 45 min on ice to stain luminal surface LAMP-1. Cells were then washed with PBS, fixed for 15 min in 4% PFA/4% sucrose, and incubated with a 488-channel secondary for 30 min. After washing, cells were permeabilized with 3% BSA/0.1% Triton X-100 for 10 min followed by a 30 min incubation in LAMP-1 primary antibody to stain total LAMP-1. Cells were incubated in a Cy3-channel secondary for 30 min then washed, mounted on slides with ProLong Diamond DAPI mounting media, and imaged on a confocal microscope at 63× (Nikon Eclipse Ti2 scanning confocal microscope).
Human brain lysates
Flash-frozen human temporal cortical brain samples from patients with incidental Lewy body disease (iLBD) and age-matched controls were acquired from the Banner Sun Health Research Institute Brain and Body Donation Program. The Institutional Review Board at UAB determined the use of human brains as not human subjects research since the tissue was from deceased persons. The tissue was sonicated in lysis buffer (50 mM Tris–HCL pH 7.4, 175 mM NaCl, and 5 mM EDTA pH 8.0) with 1% Triton X-100 and spun for 60 min at 15,000 × g to extract Triton X-100–soluble proteins. Samples were assessed for Rab27b and total protein (BlueSafe gel stain) via Western blot.
Preformed αsyn fibril assay
Preformed αsyn fibrils were prepared as previously described (Wang et al., 2018). Before use, preformed fibrils (PFFs) were diluted to 1 mg/ml and sonicated using a QSonica700 cup horn sonicator with a 16°C water bath for 15 min at a 30% amplitude with cycling 3 s on and 2 s off pulses. Fibril fragmentation of fibrils was verified using dynamic light scattering before sonicated fibrils were added to primary neuronal cultures at 1 µg/ml in neuronal media. Fibrils were added to hippocampal primary neurons at DIV 5, and cells were fixed in 4%PFA/4% sucrose/1% Triton X-100 and stained with a pS129-αsyn antibody at 7, 10, and 14 d posttreatment as previously described (Wang et al., 2018).
Western blotting
Western blotting was performed as previously described (Wang et al., 2018).
Experimental design and statistical analyses
GraphPad Prism 10 was used for statistical analysis of all experiments. All datasets were analyzed via Student's t test, one-way ANOVA with post hoc Tukey's multiple-comparison testing, or two-way ANOVA with post hoc Fisher's LSD testing. In vitro PFF experiments were analyzed by multiple unpaired t tests with correction for multiple comparisons using the Holm–Sidak method. See Table 3 for details and summary statistics.
Statistical analysis
Results
Rab27b KD promotes αsyn accumulation in lysosomes
We previously developed an inducible cell culture model that overexpresses and secretes human αsyn when exposed to doxy (Wang et al., 2018). This model is hereafter referred to as “isyn” for inducible synuclein. We knocked down Rab27b in this model using a puromycin-selected lentiviral shRNA construct and additionally generated a scrambled shRNA nt control cell line (Underwood et al., 2020). We have previously shown that KD of Rab27b in these cells exacerbates αsyn pathology and toxicity, with accumulation of Triton X-100 insoluble αsyn (Underwood et al., 2020). We also found that Rab27b KD increased levels of p62 and LC3B-positive puncta, suggesting a role in autophagic–lysosomal pathways. To examine where in the autophagic–lysosomal pathway Rab27b may act, we first performed colocalization experiments of Rab27b with proteins associated with autophagic–lysosomal pathways. We treated control isyn cells with doxy for 96 h to induce αsyn OE before staining for Rab27b in combination with the autophagosomal marker LC3B, autophagic substrate marker p62, lysosomal marker LAMP-1, early endosome marker EEA1, and late endosome marker Rab7. Uninduced cells were used as a control. We found that Rab27b partially colocalized with all markers and that its colocalization with p62 increased upon doxy induction and concurrent αsyn overexpresion (Fig. 1a–f). We further found that Rab27b colocalizes with these markers in the axons of WT mouse primary neurons (Extended Data Fig. 1-1). Additionally, we assessed the interaction of Rab27b with αsyn via PLA and found that it interacts with αsyn in isyn cells (Fig. 1g,h). This finding that Rab27b partially colocalizes with several markers in the autophagic–lysosomal pathway suggests that Rab27b may be involved in the trafficking of vesicles as they transition through this pathway.
Rab27b partially colocalizes with autophagic–lysosomal markers and interacts with αsyn. a–e, Representative images showing Rab27b colocalization with p62, LC3B, LAMP-1, EEA-1, and Rab7 in control isyn cells induced with or without doxy for 96 h. Extended Data Figure 1-1 for colocalization in primary neurons. f, Quantification of Pearson's correlation coefficients for each marker with Rab27b in individual cells. Student's t test; ***p < 0.001. g, Representative image showing PLA-positive puncta indicating instances of Rab27b interaction with αsyn with and without 96 h doxy induction. h, Control conditions for PLA include uninduced isyn cells incubated with no primary antibodies or with just a single primary antibody. Representative image showing PLA in isyn/Rab27b KD cells is also shown.
Figure 1-1
Representative images showing colocalization of Rab27b with p62, LC3b, LAMP-1, EEA1, Rab7, and αsyn in primary hippocampal neuron axons. Scale bars 10 µm. Download Figure 1-1, TIF file.
Given Rab27b's association with lysosomal structures, we next performed lysosomal fractionation to determine if αsyn accumulates within lysosomes upon Rab27b KD in isyn cells. We isolated lysosomes from doxy-induced nt-shRNA control isyn and isyn/Rab27b KD cells via ultracentrifugation and assessed the amount of αsyn found in the lysosomally enriched fractions via Western blotting. We found that αsyn levels in lysosomal fractions were higher in isyn/Rab27b KD cells compared with control cells (Fig. 2a). As previously reported (Underwood et al., 2020), baseline total lysate levels between control and isyn/Rab27b KD cells are not different. These data indicate that increased αsyn levels within the lysosomal fractions of isyn/Rab27b KD cells could be secondary to defects in αsyn lysosomal degradation.
Rab27b KD causes lysosomal impairments in inducible αsyn cells. a, Representative Western blot showing increased αsyn levels in LAMP-1–enriched fractions of isyn/Rab27b KD cells relative to control cells. Fractions 6–8 denote successive fractions, and GAPDH and histone H1 are diminished in LAMP-1 enriched fractions. αSyn levels were normalized to LAMP-1. n = 3 independent rounds. Student's t test; *p < 0.05. b, Representative images and quantification of DQ-BSA activity in nt-isyn and isyn/Rab27b KD cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; ***p < 0.001. c, Representative images and quantification of LAL activity in nt-isyn and isyn/Rab27b KD cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; ***p < 0.001. d, Top, Representative images and quantification of 10 kDa 488-dextran uptake in nt-isyn and isyn/Rab27b KD cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Two-way ANOVA; *p < 0.05; **p < 0.01. Bottom, Representative images and quantification of 10 kDa 488-dextran uptake in nt-isyn cells at 60 min under control conditions. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. One-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001. Note that the 60 m nt condition in both graphs is comprised of identical data. All error bars denote SEM.
Rab27b is required for lysosomal function in a doxy-inducible αsyn cell line
Given the accumulation of αsyn within lysosomes upon Rab27 KD in isyn cells, we next assessed lysosomal function in isyn/Rab27b KD cells. We first measured generalized lysosomal proteolysis by treating cells with an autoquenched DQ-BSA dye, which fluoresces only after exposure to active proteases. We found that isyn/Rab27b KD cells showed a 44% reduction in DQ-BSA signal relative to control cells 6 h posttreatment (Fig. 2b), indicating generalized lysosomal impairment. We then assessed the activity of the lysosomal enzyme LAL using a quenched substrate. Isyn/Rab27b KD cells showed a 46% reduction in LAL activity compared with nt-shRNA control cells (Fig. 2c). As both of these substrates must be endocytosed before lysosomal processing can occur, we next assessed the endocytic capabilities of isyn/Rab27b KD cells. We treated cells with 488-tagged 10 kDa dextran to assess bulk endocytosis and saw no differences in uptake between isyn/Rab27b KD and control cells (Fig. 2d). To validate that this methodology is capable of detecting uptake defects, we treated control cells with 3 µM CytD, an actin polymerization inhibitor shown to reduce 10 kDa dextran uptake (Ozdener et al., 2016), and saw a significant decrease in the amount of dextran internalized (Fig. 2d). We additionally treated control cells on ice, which inhibits endocytosis, and saw an almost complete loss of dextran internalization (Fig. 2d). Thus, it appears that the observed changes in lysosomal substrate unquenching are driven by lysosomal activity and not endocytic capacity.
To determine if these effects were driven by a reduction in lysosomal content, we next measured the number of lysosomes in each cell line using the lysosome-specific dye LysoTracker Red. We found no differences in the number of LysoTracker-positive vesicles in isyn/Rab27b KD cells compared with nt-shRNA control cells (Fig. 3a). To validate this, we also assessed lysosomal numbers using LAMP-1 immunocytochemistry. LAMP-1-positive vesicles were similar in nt-shRNA control and isyn/Rab27b KD cells (Fig. 3b). These data indicate that the observed impairments in lysosomal function were not due to a reduction in the number of lysosomes.
Rab27b KD does not affect lysosomal counts or pH in isyn cells. a, Representative images and quantification of LysoTracker in nt-isyn control and isyn/Rab27b KD cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. ns, nonsignificant. b, Representative images and quantification of LAMP-1–positive puncta in nt-isyn control and isyn/Rab27b KD cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. ns, nonsignificant. c, Representative images of total lysosomes (green) and acidified lysosomes (red) in nt-isyn control and isyn/Rab27b KD. As a negative control, some cells were treated with BafA to inhibit lysosomal acidification. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. ns, nonsignificant. All error bars denote SEM.
We next assessed whether lysosomal pH was altered upon Rab27b KD in isyn cells. Living isyn/Rab27b KD and control cells were induced with doxy for 96 h before being stained and quantified for both total and acidified lysosomes. A subset of cells were treated with BafA to inhibit acidification as a negative control. Isyn/Rab27b KD cells showed a similar proportion of acidified lysosomes relative to the total number compared with nt-shRNA control isyn cells (Fig. 3c).
We then examined the activity and expression of the lysosomal enzymes Ctsd and GCase. Both of these enzymes are implicated in PD; Ctsd is the key lysosomal cathepsin thought to degrade αsyn (Cullen et al., 2009; Prieto Huarcaya et al., 2022), and mutations in the GBA1 gene increase PD risk (Do et al., 2019). We found that Rab27b KD in isyn cells reduced Ctsd and GCase activities compared with nt-shRNA control isyn cells (Fig. 4a,b). We next assessed levels of immature and mature Ctsd and GCase levels in these cells by Western blot. Isyn/Rab27b KD cells showed a reduction in the expression of both mature Ctsd and mature, glycosylated GCase compared with control cells (Fig. 4c; Extended Data Fig. 4-1).
Lysosomal enzymes are impaired by Rab27b KD. a, Quantification of Ctsd activity in nt-isyn and isyn/Rab27b KD cells. n = 4 independent rounds. Student's t test; ****p < 0.0001. b, Quantification of GCase activity in nt-isyn and isyn/Rab27b KD cells. n = 6 independent rounds. Student's t test; ***p < 0.001. c, Representative Western blot and quantification of immature and mature Ctsd and GCase levels in nt-isyn and isyn/Rab27b KD cells. n = 3 independent rounds. Student's t test; *p < 0.05; **p < 0.01. Extended Data Figure 4-1 for full blots. All error bars denote SEM.
Figure 4-1
Full-size representative Western blots showing Ctsd and Gcase expression in isyn/Rab27b KD and nt-isyn control cells. Download Figure 4-1, TIF file.
After synthesis in the endoplasmic reticulum (ER), hydrolytic lysosomal enzymes are trafficked to the Golgi apparatus where they undergo maturation and are tagged with mannose-6-phosphate (M6P) prior to sorting into early endosomes and lysosomes (Lakpa et al., 2021; Meras et al., 2022). To determine if the reduced lysosomal enzyme activities were secondary to a reduction in enzyme localization within lysosomes, we assessed the colocalization of Ctsd with LAMP-1–positive puncta in isyn/Rab27b KD and control cells. We found reduced colocalization of Ctsd with LAMP-1 in isyn/Rab27b KD cells, potentially indicating a defect in enzymatic trafficking (Fig. 5a). We assessed GCase enzyme maturation using an EndoH assay. Lysosomal hydrolases undergo N-glycosylation in the ER before being trafficked to the Golgi. Resistance to EndoH develops as lysosomal hydrolases further mature within the Golgi (Cuddy and Mazzulli, 2021). We treated lysates from nt-shRNA control and isyn/Rab27b KD cells with and without EndoH and then examined the resulting GCase banding patterns by Western blot (Fig. 5b). We observed similar levels of sensitivity to EndoH in isyn/Rab27b KD cells compared with control cells as noted by the ratio of EndoH-resistant/Endo H-sensitive GCase bands (Fig. 5b). This suggests that any trafficking defects in lysosomal hydrolases would occur distal to the Golgi. Overall, isyn/Rab27b KD cells exhibited defects in lysosomal enzymatic activity, maturation, and localization but no changes in lysosomal counts or lysosomal pH.
Rab27b KD changes Ctsd localization but does not impact GCase EndoH sensitivity. a, Representative images and quantification showing colocalization of Ctsd with LAMP-1 in isyn/Rab27b KD and nt-isyn control cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; *p < 0.05. b, Representative Western blot and quantification of EndoH-sensitive/EndoH-resistant GCase in isyn/Rab27b KD compared with nt-isyn control cells. n = 3 independent rounds. Student's t test; ns, nonsignificant.
Lysosomal activity is impaired in the brains of Rab27b KO mice
We next examined lysosomal function in primary neurons from Rab27b KO mice. We generated Rab27b single KO mice after crossing Rab27a/Rab27b DKO mice obtained from Dr. Seabra (Tolmachova et al., 2007) with WT C57BL/6J mice. Of note, we previously found that the Rab27a/b DKO mouse carries a spontaneous deletion that includes Snca, the gene that encodes αsyn, and Mmrn1, the gene that encodes multimerin 1 (Pattanayak et al., 2022). This deleted region on chromosome 6 was restored in a subset of progeny after crossing the DKO mice with C57BL/6J mice. Mice heterozygous for Rab27a and Rab27b that lacked the deletion were selected for subsequent breeding in order to generate Rab27b single KO mice (Fig. 6a). We confirmed that this previously deleted region, including the Snca locus, was present in the Rab27b single KO mice (Fig. 6b). Interestingly, in our attempts to generate Rab27a/Rab27b DKO mice without the deletion, we found that KO of both Rab27a and Rab27b was not compatible with the presence of the Snca/Mmrn1 region. Only three Rab27a/b DKO mice were born out of a total of 97 pups born from mating Rab27a−/− Rab27b −/− αsyn+/− mice to each other, which was much less than the expected 25% (Table 4). We attempted three additional crosses to generate the Rab27 DKO mouse line without the Snca/Mmrn1 deletion, yet these crosses also primarily produced fewer-than-expected Rab27 DKO pups (Table 4).
Rab27b KO primary neurons display lysosomal deficits but no changes in lysosomal number or uptake capabilities. a, Schematic depicting Rab27b KO breeding strategy. b, Rab27b KO line genotyping validating that Rab27b KO and littermate WT mice express the Snca locus. c, Representative images and quantification of DQ-BSA activity in WT and Rab27b KO neurons. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. ***p < 0.001. d, Representative images and quantification of LAL activity in WT and Rab27b KO neurons. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; **p < 0.01. e, Representative images and quantification of LysoTracker-positive puncta in WT and Rab27b KO neurons. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; ns, nonsignificant. f, Representative images and quantification of 10 kDa 488-dextran uptake in WT and Rab27b KO neurons at 30 and 60 min. The signal is quantified per frame and averaged per independent assay. n = 3 independent rounds. Two-way ANOVA. *p < 0.05. Extended Data Figure 6-1 for CytD and ice uptake control images. All error bars denote SEM.
Figure 6-1
Representative images showing reduced uptake of 10 kDa 488-dextran in WT primary hippocampal neurons treated with 6 µM Cytochalasin D or treated on ice. Download Figure 6-1, TIF file.
Crosses to generate Rab27 DKO mice result in lower-than-expected proportions of DKO mice
Once we established the single Rab27b KO mouse line, we first examined lysosomal function in primary hippocampal neurons at DIV 8. Lysosomal proteolytic activity as assessed by DQ-BSA cleavage was reduced by 44% in neurons from Rab27b KO mice compared with WT (Rab27b+/+) littermate controls (Fig. 6c). LAL activity was similarly reduced in Rab27b KO neurons compared with WT neurons (Fig. 6d). Lysosomal numbers as determined by LysoTracker-positive counts were not different between WT and Rab27b neurons (Fig. 6e). To validate that these findings were driven by lysosomal activity and not confounded by differences in the uptake of quenched substrates, we again assessed bulk endocytosis in Rab27b KO and WT neurons using 488-tagged dextran (10 kDa). Dextran uptake was comparable between WT and Rab27b KO neurons (Fig. 6f), and both CytD (6 µM) and ice controls inhibited uptake (Extended Data Fig. 6-1).
We also examined whether increased αsyn load in primary neurons would exacerbate the lysosomal defects in primary hippocampal cultures. We crossed Rab27b KO mice with the transgenic A53T human αsyn mouse (Lee et al., 2002; Fig. 7a). Western blotting for αsyn in these animals confirmed significantly elevated αsyn in both A53T+ genotypes relative to A53T− controls (Fig. 7b). Lysosomal proteolytic activity as measured by DQ-BSA cleavage was reduced in Rab27b−/− A53T-negative (Rab27b KO/A53T−) and Rab27b−/− A53T hemizygous (Rab27b KO/A53T+) neurons compared with either Rab27b+/+ A53T-negative (WT) or Rab27b+/+ A53T hemizygous (A53T+) neurons, yet there was no additional impairment of DQ-BSA cleavage in Rab27b KO/A53T+ neurons compared with Rab27b KO neurons (Fig. 7c). Similarly, LAL activity was significantly reduced in both Rab27b KO and Rab27b KO/A53T+ neurons, but there was no difference between Rab27b KO and Rab27b KO/A53T+ neurons (Fig. 7d).
Rab27b KO × A53T hemizygous mice do not display exacerbated lysosomal pathology. a, Schematic depicting Rab27b KO × A53T hemizygous breeding strategy. b, Western blot and quantification showing elevated αsyn in A53T hemizygous mice at P8. n = 3 mice/group; one-way ANOVA with Tukey's multiple-comparison test; **p < 0.01. c, Representative images and quantification of LAL activity in WT, A53T hemizygous, Rab27b KO, and Rab27b KO × A53T hemizygous neurons. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. One-way ANOVA with Tukey's multiple-comparison test; ***p < 0.001; ****p < 0.0001. d, Representative images and quantification of DQ-BSA activity in WT, A53T hemizygous, Rab27b KO, and Rab27b KO × A53T hemizygous neurons. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. One-way ANOVA with Tukey's multiple-comparison test; ****p < 0.0001. All error bars denote SEM.
To examine if lysosomal enzymatic defects were also present in vivo, we measured Ctsd and GCase activity in cortical and hippocampal brain lysates from 2–4-month-old WT and Rab27b KO mice. Ctsd activity was reduced in both the cortex and hippocampus of Rab27b KO mice compared with WT littermates (Fig. 8a). Similarly, GCase activity was reduced in the cortex and hippocampus of Rab27b KO mice compared with WT mice (Fig. 8b; Extended Data Fig. 8-1). We found a decrease in glycosylated GCase protein levels in both the hippocampus and cortex, but did not detect alterations in the levels of Ctsd (Fig. 8c,d). We then assessed Ctsd colocalization with LAMP-1 in Rab27b KO primary neurons compared with WT controls and found reduced colocalization in Rab27b KO neurons, again indicating a potential defect in enzymatic trafficking to lysosomes (Fig. 8e).
Rab27b KO mice show impaired lysosomal enzyme activity, expression, and localization. a, Quantification of Ctsd activity in cortical and hippocampal lysates from 2–4-month-old WT and Rab27b KO mice. n = 6 mice/group. Student's t test; ****p < 0.0001. b, Quantification of GCase activity in cortical and hippocampal lysates from 2–4-month-old WT and Rab27b KO mice. n = 6 mice/group. Student's t test; **p < 0.01. c, d, Representative Western blots and quantification of Ctsd or GCase levels in hippocampal (c) and cortical (d) lysates from 2–4-month-old WT and Rab27 KO mice. n = 3–4 mice/group. Student's t test; *p < 0.05; **p < 0.01. Extended Data Figure 8-1 for full blots. e, Representative images and quantification of Ctsd colocalization with LAMP-1 in primary hippocampal neurons from Rab27b KO and WT mice. Pearson's correlation coefficient is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test; *p < 0.05. All error bars denote SEM.
Figure 8-1
Full-size Western blots showing Ctsd and Gcase expression in Rab27b KO and WT mice. Download Figure 8-1, TIF file.
Rab27b KO exacerbates αsyn pathology
We next examined whether Rab27b KO was sufficient to cause increases in insoluble αsyn in the brain tissue. We measured Triton X-100–soluble and insoluble fractions from the hippocampus and cortex of Rab27b KO and WT littermates at 12–15 months of age. αSyn levels in both the Triton X-100 soluble and insoluble fractions were comparable in WT and Rab27b KO lysates (Fig. 9a,b). We validated our fractionation by assessing pS129 phosphorylated αsyn and found it was only present in Triton X-100 insoluble fractions (Extended Data Fig. 9-1). We also assessed the autophagic substrate marker p62 and found no changes in its expression in 12–15-month-old mice in either Triton X-100 soluble or insoluble fractions (Fig. 9c,d). We did not see any differences in LAMP-1 protein levels in WT versus Rab27b KO brain lysates (Fig. 9c,d).
Rab27b KO mice show no changes in autophagic–lysosomal markers or αsyn solubility. a, b, Representative Western blots and quantifications of αsyn levels in Triton X-100–soluble (a) and Triton X-100–insoluble (b) cortical and hippocampal lysates from 12–15-month-old WT and Rab27b KO mice. n = 6 mice/group. Student's t test. ns, nonsignificant. Extended Data Figure 9-1 for sample pS129 αsyn and GAPDH validation blots. c, d, Representative Western blots and quantifications of Triton X-100 soluble p62 and LAMP-1 and Triton X-100 insoluble p62 expression in cortical (c) and hippocampal (d) lysates from 12–15-month-old WT and Rab27b KO mice. n = 6 mice/group. Student's t test. ns, nonsignificant. All error bars denote SEM.
Figure 9-1
a-b: Sample Western blot showing that pS129-αsyn is only present in Triton X-100 insoluble mouse brain fractions. c: Sample Western blot showing less GAPDH in Triton X-100 insoluble brain fractions compared to Triton X-100 soluble brain fractions. Download Figure 9-1, TIF file.
We then examined Rab27b KO in the context of αsyn stress by assessing its effect on αsyn aggregation induced by αsyn PFFs in culture. Primary hippocampal neurons from WT and Rab27b KO littermates were treated with αsyn PFFs at 1 µg/ml at DIV 5, and then cultures were fixed and stained for Triton X-100–insoluble phosphorylated αsyn at S129 (psyn) as a measure of aggregated αsyn as previously described (Wang et al., 2018). Soluble αsyn was digested with 1% Triton X-100 during fixation. We found that Rab27b KO primary neurons accumulated more insoluble psyn at 7 and 10 d post-PFF treatment (Fig. 10), indicating that KO of Rab27b can exacerbate αsyn-induced pathology.
Rab27b KO causes increased accumulation of insoluble pS129 αsyn upon PFF treatment. Representative images and quantification showing pS129 immunostaining of WT and Rab27b KO hippocampal primary neurons over time after treatment with either monomeric or fibrillar αsyn. Points represent individual coverslips across six independent rounds. Unpaired Student's t tests with Holm–Sidak correction for multiple comparisons; *p < 0.05.
Rab27b KO impairs anterograde lysosomal trafficking in axons
Rab27b has been implicated in vesicular trafficking in axons (Arimura et al., 2009). To test if Rab27b KO could affect trafficking of acidic vesicles labeled with LysoTracker in axons, we performed live imaging of WT and Rab27b KO hippocampal neurons at DIV 8 (Fig. 11a,b). We found that the percentage and the density of cargo moving in the anterograde direction was significantly decreased in Rab27b KO neurons compared with WT (Fig. 11c,d). Additionally, the percentage of LysoTracker-positive vesicles that were stationary was increased in Rab27b KO neurons compared with WT (Fig. 11c). We also found that the net velocity of acidic vesicles was significantly reduced in both anterograde and retrograde directions in Rab27b KO neurons compared with WT (Fig. 11e). Although there was an increase in stationary tracks, the pause duration of LysoTracker-positive vesicles was not affected (Fig. 11f). These data suggest that Rab27b regulates anterograde trafficking of acidic vesicles within neurons.
Rab27b KO impairs axonal acidic vesicle trafficking in primary neurons. a, b, Representative images and kymographs depicting the movements of LysoTracker-positive puncta in WT (a) and Rab27 KO (b) primary neurons. c–f, Quantification of the percentage of net cargo population, the number of tracks per micrometer (density), net velocity, and pause duration of LysoTracker-positive vesicles in WT and Rab27 KO primary neurons. Points represent individual tracks from six neurons per genotype measured across four independent rounds. Shapiro–Wilk normality testing followed by either Student's t tests or Mann–Whitney U tests (see Table 3 for details); *p < 0.05; **p < 0.01; ****p < 0.001. All error bars denote SEM.
Rab27b KD impairs lysosomal exocytosis
Anterograde lysosomal trafficking is important for lysosomal exocytosis, which is an important mechanism for protein clearance that has been found to protect against αsyn toxicity (Tsunemi et al., 2019). We therefore assessed lysosomal exocytosis in our isyn/Rab27b KD and control cell lines. We stimulated lysosomal exocytosis with BafA before using a luminal LAMP-1 antibody to detect the residual LAMP-1 left on the surface of the plasma membrane following lysosomal fusion. We found that isyn/Rab27b KD cells had significantly reduced surface LAMP-1 signal, indicating a reduction in lysosomal exocytosis (Fig. 12). We have previously shown that KD of Rab27b in isyn cells reduces the amount of αsyn secreted into the culture media (Underwood et al., 2020), which may be driven by reduced lysosomal exocytic capacity.
Rab27b KD impairs lysosomal exocytosis. Representative images and quantification showing surface LAMP-1 staining in isyn/Rab27b KD compared with nt-isyn control cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. *p < 0.05.
Rab27b OE enhances lysosomal enzymatic function
We next determined if OE of Rab27b could enhance lysosomal activity. Isyn cells were transduced with either a GFP lentivirus or GFP-tagged Rab27b lentivirus which were both selectable by puromycin. We confirmed increased Rab27b expression in isyn cells transduced with GFP-tagged Rab27b lentivirus compared with GFP lentivirus (Fig. 13a). DQ-BSA cleavage was increased by 23% in isyn/Rab27b OE cells compared with isyn/GFP cells (Fig. 13b). Similarly, Ctsd and GCase enzyme activities were increased by 36% and 15%, respectively, in isyn/Rab27b OE cells compared with isyn/GFP cells (Fig. 13c,d). We did not observe any changes in LAMP-1–positive vesicles between isyn/Rab27b OE cells and isyn/GFP cells (Fig. 13e), but we did detect increases in both mature Ctsd and GCase protein levels (Fig. 13f; Extended Data Fig. 13-1).
Rab27b OE cells display reduced αsyn pathology and enhanced lysosomal activity. a, Representative image showing immunostaining for Rab27b in isyn/GFP control and isyn/Rab27b OE cells. b, Representative images and quantification of DQ-BSA activity in isyn/GFP control and isyn/Rab27b OE cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. *p < 0.05. c, Quantification of Ctsd activity in isyn/GFP control and isyn/Rab27b OE cells. n = 3 independent rounds. Student's t test; ***p < 0.001. d, Quantification of GCase activity in isyn/GFP control and isyn/Rab27b OE cells. n = 3 independent rounds. Student's t test; *p < 0.05. e, Representative images and quantification of LAMP-1–positive puncta in isyn/GFP control and isyn/Rab27b OE cells. The signal is quantified per cell and averaged per independent assay. n = 3 independent rounds. Student's t test. f, Representative Western blots and quantification of Ctsd and GCase levels in isyn/GFP control and isyn/Rab27b OE cells. n = 5 independent rounds. Student's t test. *p < 0.05; **p < 0.01. Extended Data Figure 13-1 for full blots. g, Representative Western blot and quantification of αsyn levels in Triton X-100–soluble and Triton X-100–insoluble cell lysates from isyn/GFP control and isyn/Rab27b OE cells. n = 4 independent rounds. Student's t test; *p < 0.05. h, Representative Western blot and quantification of αsyn levels in LAMP-1–enriched fractions of isyn/GFP control and isyn/Rab27b OE cells. Fractions 1–6 denote successive fractions. GAPDH and histone H1 are depleted in LAMP-1–enriched fractions. αSyn levels are normalized to LAMP-1. n = 4 independent rounds. Student's t test; *p < 0.05. i, Representative Western blot and quantification of baseline levels of αsyn in input controls of induced isyn/Rab27b OE and isyn/GFP control cells. Student's t test. ns, nonsignificant. j, Representative Western blot and quantifications of αsyn in the lysate and secreted into the conditioned culture media (CM) of isyn/Rab27b OE and isyn/GFP control cells. n = 3 independent rounds. Student's t test; **p < 0.01. All error bars denote SEM.
Figure 13-1
Full-size representative Western blots showing Ctsd and Gcase expression in isyn/Rab27b OE and isyn/GFP control cells. Download Figure 13-1, TIF file.
Rab27b OE is protective against αsyn pathology
We previously determined that Rab27b KD in isyn cells results in increased intracellular Triton X-100–insoluble αsyn (Underwood et al., 2020). To determine if Rab27b OE could reduce insoluble αsyn accumulation, we prepared Triton-soluble and insoluble protein fractions from doxy-induced isyn/Rab27b OE and isyn/GFP cells. αSyn levels in the Triton X-100–soluble fractions were not significantly changed between isyn/Rab27b OE and isyn/GFP cells, but Rab27b OE significantly reduced Triton X-100–insoluble αsyn levels by 65% compared with control isyn/GFP cells (Fig. 13g).
Given the reduction in insoluble αsyn with Rab27b OE, we next assessed the levels of αsyn retained in lysosomal fractions. We found that induced isyn/Rab27b OE cells show reduced αsyn in lysosomally enriched fractions compared with induced isyn/GFP cells, potentially indicating enhanced αsyn lysosomal degradation with Rab27b OE (Fig. 13h). Of note, total cellular lysate levels of αsyn in isyn/GFP versus isyn/Rab27b OE cells did not differ in unfractionated input controls (Fig. 13i).
We finally assessed the release of αsyn into the culture media in isyn/Rab27b OE compared with isyn/GFP control cells and found reduced αsyn release, though there was again no change in total lysate αsyn levels (Fig. 13j). Overall, isyn cells with Rab27b OE display enhanced lysosomal activity and reduced αsyn accumulation, indicating a potentially protective role of Rab27b against αsyn pathology.
Rab27b is elevated in human iLBD
We have previously shown that Rab27b is elevated in temporal cortical lysates from human DLB subjects relative to age-matched controls (Underwood et al., 2020). We evaluated whether Rab27b protein levels were altered at preclinical stages by measuring Rab27b levels in a cohort of iLBD subjects. iLBD is viewed to represent the earliest stages of synucleinopathy and is pathologically defined by the presence of Lewy bodies or neurites in autopsied subjects without clinical diagnosis of Parkinsonism or dementia (DelleDonne et al., 2008; Dickson et al., 2008; Dijkstra et al., 2014; Iacono et al., 2015). We found a 55% increase in Rab27b levels in temporal cortical lysates from iLBD patients relative to age-matched controls (Fig. 14). Based on these findings, we conclude that the increase in Rab27b levels we have previously observed in DLB and PD subjects is likely a compensatory mechanism that occurs early in disease.
Rab27b is elevated in human iLBD. Representative Western blot showing Rab27b levels and corresponding total protein per lane. Quantification of Rab27b levels in human temporal cortical lysates from iLBD subjects relative to age-matched controls. n = 45/group. Student's t test; ****p < 0.0001. Error bars depict SEM.
Discussion
Our findings point to a critical role for Rab27b in lysosomal function and the clearance of αsyn. We had previously shown that Rab27b promoted αsyn aggregation and toxicity using a doxy-inducible αsyn paracrine cell model. Here, we show that Rab27b KD disrupted lysosomal function, which resulted in diminished αsyn clearance as demonstrated by increased αsyn levels in lysosomes. Similarly, we found lysosomal defects in primary neuronal cultures and in the cortex and hippocampus of Rab27b KO mice. Rab27b KO neurons showed increased αsyn pathology upon αsyn PFF treatment compared with WT neurons. We also observed increased Rab27b protein levels in temporal cortical lysates from subjects with iLBD. This suggests that increases in Rab27b may reflect compensatory mechanisms to promote lysosomal function in the brain in order to handle αsyn pathological stress. Indeed, we found that Rab27b OE enhanced lysosomal function in inducible αsyn cells and reduced levels of insoluble αsyn.
How Rab27b specifically enhances lysosomal function in neurons is still not fully clear but our data suggests that Rab27b regulates the maturation and transport of lysosomal enzymes to their ultimate destination in lysosomes. We observed a reduction in mature Ctsd and GCase protein levels with Rab27b KD in isyn cells and an increase in these mature enzymes with Rab27b OE in isyn cells. Lower levels of higher molecular weight GCase were also observed in Rab27b KO mouse brains, although no clear difference was seen in Ctsd. Differences in the ability of the Ctsd antibody used to detect different Ctsd species in mice versus human cell types could account for this discrepancy. The general reduction in mature lysosomal hydrolase levels suggests that Rab27b may regulate the transport of these enzymes to lysosomes. Upon synthesis in the ER, lysosomal enzymes are trafficked to the Golgi where they undergo maturation with glycosylation changes and become tagged with M6P, which then allows for sorting into small vesicles toward early endosomes and lysosomes. Consistent with this effect, we observed reduced colocalization of Ctsd with LAMP-1–positive vesicles both in isyn cells and in primary neurons upon Rab27b KD/KO. Our EndoH sensitivity assay suggests that the trafficking defect occurs distal to the Golgi apparatus, since control and Rab27b KD cells showed similar levels of sensitivity to EndoH. Based on these data, we hypothesize that Rab27b supports the transport of M6P receptor-positive vesicles containing lysosomal enzymes from the Golgi to lysosomes where they are cleaved and activated. Future studies will examine the exact subcellular localization at which Rab27b acts in the transport of lysosomal hydrolases.
Additionally, we found that Rab27b KO neurons showed disruptions in the anterograde transport of LysoTracker-positive vesicles within axons, suggesting that Rab27b is involved in trafficking of lysosomes to appropriate locations within the cell. The role of Rab27b in axonal transport is not entirely novel. For example, Rab27b has been implicated in the axonal transport of TrkB-positive vesicles (Arimura et al., 2009). The role of Rab27b in axonal transport could have significant impact on αsyn clearance. Effective protein clearance is dependent on bidirectional transport of autophagic vesicles and lysosomes in neurons. αSyn protein levels are enhanced at synapses, and evidence points to the initiation of αsyn pathology at the axon terminal (Volpicelli-Daley et al., 2011; Uchihara, 2017; Wong et al., 2019; Schaser et al., 2020), where degradative capacity is limited (Ferguson, 2018; Farfel-Becker et al., 2019; Roney et al., 2022). Protein clearance at distal terminals is dependent on the retrograde transport of autophagosomes that engulf misfolded proteins to the soma where they fuse with mature lysosomes (Ferguson, 2018; Kulkarni et al., 2022; Roney et al., 2022). Protein clearance is also dependent on the delivery of lysosomes to distal axons where local fusion with autophagic vesicles to initiate clearance occurs (Farfel-Becker et al., 2019; Roney et al., 2022). Indeed, disruption of vesicular transport within axons has been shown to reduce αsyn clearance (Farfel-Becker et al., 2019).
Our data also reveal that Rab27b facilitates lysosomal exocytosis, pointing to another role of Rab27b in lysosome trafficking in the cell. We found that Rab27b KD reduced lysosomal exocytosis in isyn cells, which is consistent with previous findings demonstrating regulation of lysosomal exocytosis by Rab27b in oligodendrocytes (Shen et al., 2016). Lysosomal exocytosis has been implicated in synucleinopathies (Xie et al., 2022; Abe et al., 2024). This release of αsyn via lysosomal exocytosis may be a mechanism by which cells clear unwanted αsyn, and increased lysosomal exocytosis has been found to protect against αsyn toxicity (Tsunemi et al., 2019). Reduced lysosomal exocytosis resulting from Rab27b KD may therefore contribute to the exacerbated αsyn pathology seen in isyn cells.
We have previously shown that Rab27b KD reduces the secretion of αsyn into the culture media (Underwood et al., 2020). This may be driven by a loss of lysosomal exocytic capacity; Rab27b KD cells may be unable to secrete excess αsyn, and this may contribute to the exacerbated αsyn pathology seen in that cell line. Surprisingly, we found that OE of Rab27b also reduces the amount of αsyn secreted into the culture media. This may result from an enhanced ability to degrade αsyn; if Rab27b OE facilitates lysosomal function and increases degradative capacity, the cells may be degrading rather than secreting excess αsyn.
We previously found that Rab27b protein levels are increased in human PD and DLB brains. Here we provide additional data showing that Rab27b protein levels are also increased in iLBD brains. iLBD is viewed as a preclinical form of PD and DLB in which patients have evidence of Lewy body pathology yet have not developed clear clinical signs of synucleinopathy (DelleDonne et al., 2008; Dickson et al., 2008; Dijkstra et al., 2014; Iacono et al., 2015). This finding that Rab27b is elevated at this preclinical stage suggests that increases in Rab27b reflect compensatory mechanisms by which neurons are trying to handle excess proteostatic stress from αsyn aggregates. Further boosting of Rab27b function could provide a therapeutic target for PD and DLB. This may be most practical in GBA-related disorders, in which GCase activity is diminished and thought to be responsible for the increased risk in synucleinopathies. Given our findings that Rab27b OE can boost GCase activity, mechanisms targeting Rab27b could be a therapeutic intervention for GBA.
Our finding that lysosomal function was diminished in Rab27b KO neurons even in the absence of excess αsyn suggests that an endogenous function for Rab27b in neurons is maintenance of lysosomal activity. This finding suggests that Rab27b could play a role in other neurodegenerative disorders marked by protein aggregation, such as Alzheimer's disease, Huntington's disease, and others. Evaluation of the impact of Rab27b is important to assess in other neurodegenerative disorders.
In conclusion, we found that loss of Rab27b reduces lysosomal function in neuronal cell lines, primary neurons, and in brain tissue. Rab27b OE increases lysosomal function and reduces αsyn accumulation, pointing to Rab27b as a target for therapeutic intervention.
Footnotes
- Received August 20, 2024.
- Revision received February 4, 2025.
- Accepted February 4, 2025.
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We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City (DelleDonne et al., 2008; Dickson et al., 2008; Dijkstra et al., 2014; Iacono et al., 2015) for the provision of human biological materials. The Brain and Body Donation Program has been supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG019610 and P30AG072980, Arizona Alzheimer’s Disease Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901, and 1001 to the Arizona Parkinson's Disease Consortium), and the Michael J. Fox Foundation for Parkinson’s Research. This study was supported by NIH [RF1NS115767 (T.A.Y.); P50 NS108675 (T.A.Y); T32 5T32GM135028 (K.S.); T32 5T32NS095775 (K.S.); F31 1F31NS135928-01A1 (K.S.)] and the American Parkinson Disease Association. Mouse breeding graphics were generated using BioRender.com.
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↵*K.S. and R.P. are co-first authors.
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The authors declare no competing financial interests.
- Correspondence should be addressed to Talene A. Yacoubian at tyacoubian{at}uabmc.edu.
- Copyright © 2025 the authors
