The Parkinson’s disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Background Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson’s disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results We first identified specific non-coding variants in CTSB that drive the association with PD and are linked to changes in brain CTSB expression levels. Using iPSC-derived dopaminergic neurons we then find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. Moreover, in cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. Similarly, in midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition or knockout potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions The results of our genetic and functional studies indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.


Introduction
Parkinson's disease (PD) is characterized by both the degeneration of dopaminergic neurons in the substantia nigra and by the accumulation of Lewy bodies; proteinaceous inclusions composed in largely of misfolded and aggregated α-synuclein (α-syn) 1 .Mutations that increase protein levels of α-syn or its propensity to aggregate contribute substantial genetic risk to PD 2,3 , supporting the predominant hypothesis that α-syn aggregation is a key step in the pathological cascade leading to neurodegeneration in PD.The lysosome serves as the principal site for degradation of aggregated α-syn [4][5][6] , and mutations in lysosomal genes also represent a substantial genetic risk for PD 7 .Thus, there is great interest in understanding the lysosomal pathways that mediate α-syn clearance.Cathepsin B (catB, encoded by the CTSB gene) is a proteolytic enzyme of the cysteine cathepsin family with endo-and exo-peptidase activity that is normally localized to the lysosomal lumen 8 .CatB has been implicated both in the lysosomal degradation of α-syn and as a genetic risk factor for PD.In the present study we further elucidate the relationship between CTSB variants and PD risk and demonstrate that catB modulates lysosome function and the clearance of α-syn aggregates in cell lines and human dopaminergic neurons.
The importance of lysosomal function in PD is well established by both functional and genetic studies 7 .Recently, genome-wide association studies (GWAS) have identi ed signi cant association between variants in the CTSB genetic locus and the risk of PD generally 9 and speci cally in carriers of pathogenic GBA1 variants 10 .In addition to genetic evidence linking CTSB to PD, catB protein or activity levels are reduced in several cellular models of PD.For example, pathological α-syn species have been shown to impair catB tra cking to the lysosome 11 , while iPSC derived neurons harboring mutations in SNCA or GBA1 exhibited reduced catB activity 12,13 .Additionally, knockout of the PD risk gene TMEM175 impairs catB activity by destabilizing lysosome pH 14,15 , while mutations in LRRK2, the most common cause of familial PD, have been shown to suppress catB expression or activity in the lysosome 16,17 .Thus, several lines of evidence suggest that disrupted catB function could play a role in PD pathogenesis.
One potential mechanism linking catB to PD is through its ability to cleave both monomeric and aggregated forms of α-syn, which has been demonstrated in vitro [18][19][20] .However, while this could argue for a protective role of catB against synucleinopathy, the α-syn truncations produced by in vitro catB cleavage exhibit an increased propensity to aggregate 21 and although lysosome function is essential for degradation of brillar α-syn 22 , it has also been suggested that catB activity contributes to α-syn toxicity in some cellular models 23 .Moreover, catB has been linked to the α-syn dependent activation of in ammatory pathways 24 and is a key regulator of cell death in many cellular contexts 25 .Thus, there are compelling arguments to be made in favor of both protective and potentially pathogenic actions of catB in the etiology of PD and its speci c role remains to be elucidated.
Here, we aim to both clarify the genetic evidence pertaining to how CTSB variants may contribute to PD etiology, and to functionally characterize the role of catB in relation to lysosome function and α-syn clearance.We rst provide genetic evidence that PD-associated CTSB variants decrease expression levels of the enzyme.Second, by pharmacologically and genetically modulating catB expression or activity in cell lines and human dopaminergic neurons we demonstrate that catB is required for lysosomal functions including glucocerebrosidase activity and contributes to clearance of brillar α-syn.These ndings argue in favor of a protective effect of catB in PD.

Fine mapping and eQTL analysis of CTSB variants
To identify the most likely variant driving the PD-association in the CTSB locus, we performed analyses using the summary statistics from the most recent PD GWAS 9 and multiple bioinformatic tools.First, to examine whether there are multiple independent associations in this locus, we used genome-wide complex trait conditional and joint analysis (GCTA-COJO) 26 , using default parameters.For downstream analyses, we generated a linkage disequilibrium (LD) matrix for the CTSB locus using PLINK 1.9 27 , including all variants within ± 1Mbp from the top variant.Then, we performed ne-mapping of the CTSB locus to nominate the most likely driving variants using FINEMAP 28 , with minor allele frequency (MAF) threshold > 0.01.Expression quantitative trait locus (eQTL) analysis was performed using colocalization (COLOC) 29 , which examines whether the same variants associated with the trait (PD) are also associated with gene expression.QTLs were tested in a total of 109 tissues and cells (Supplementary Table 1) To further explore the link between genetic variants, QTLs and PD we used Summary-data-based Mendelian Randomization (SMR), which uses Mendelian randomization to suggest potential causality, followed by heterogeneity in dependent instruments (HEIDI) to distinguish between pleiotropy (or causality) and LD 30 .

Rare Variant Analysis
Rare variant analysis was performed on 5,801 PD cases and 20,427 controls across six cohorts (Supplementary Table 2).All patients were diagnosed by movement disorder specialists according to the UK brain bank criteria 31 or MDS diagnostic criteria 32 .From the AMP-PD and UKBB cohorts we only included participants of European ancestry and excluded any rst and second-degree relatives from the analysis.Quality control procedures for AMP-PD and UKBB were performed as previously described in detail 33,34 .
In addition, we conducted sequencing on four distinct cohorts at McGill University (McGill cohort, Columbia cohort, Sheba Medical Center cohort and Pavlov and Human Brain Institutes cohort).We performed sequencing of the CTSB gene, including exon-intron boundaries (± 50bps) and the 5' and 3' untranslated regions (UTRs) using molecular inversion probes (MIPs) as described previously 35 .The full protocol is available at https://github.com/gan-orlab/MIP_protocol.Library sequencing was performed by the Genome Quebec Innovation Centre on the Illumina NovaSeq 6000 SP PE100 platform.We used Genome Analysis Toolkit (GATK, v3.8) for post-alignment quality checking and variant calling 36 .We applied standard quality control procedures 37 .In brief, only variants with minor allele frequency (MAF) of less than 1% and a minimum quality score (GQ) of 30 were included in the analysis.The average coverage for CTSB in cohorts sequenced at McGill was > 4000X with 95% nucleotides covered at 30x (Supplementary Table 3).
To analyze rare variants, we applied the optimized sequence Kernel association test (SKAT-O, R package) 38 with further meta-analysis between the cohorts using metaSKAT package 39 .We performed separate analyses for the whole gene, non-synonymous and functional (nonsynonymous, stop and frameshift variants) and variants with Combined Annotation Dependent Depletion (CADD) scores of ≥ 20 40 .We adjusted for sex, age and ethnicity in all analyses.We also analyzed whether rare CTSB variants affected age at onset of PD.

Generation of CTSB-KO and SNCA-KO iPSC
The previously characterized AIW002-02 iPSC cell line 41 was used to generate CTSB and SNCA knockout lines.CRISPR gRNAs were designed using Synthego and the sequences of reagents used are depicted in Supplementary Table 4.The SNCA-KO line was created by using two gRNAs to introduce a 122bp deletion into exon 1 of the gene.The gRNA sequences were cloned into a Cas9/puromycin expression vector PX459 (Addgene, #48139) and transfected into iPSCs using Lipofectamine TM stem reagent (ThermoFisher Scienti c).Transfected iPSCs were selected in 0.3µg/mL puromycin for 72h and surviving colonies were manually picked and expanded for PCR screening to con rm deletion of the target region.Colonies con rmed to be knockout by PCR screening were further validated by sanger sequencing, and loss of protein was con rmed by Western blot in differentiated neurons.
The CTSB-KO cell line was created by HDR using a single gRNA and ssDNA repair template to introduce a stop tag in exon 4 of the gene.Cas9 nuclease, gRNA and a ssDNA repair template for HDR were introduced by Lonza Nucleofection.Edited alleles were detected with ddPCR to select edited clones and deletion was veri ed by PCR screening followed by sanger sequencing, and nally loss of protein was determined by Western blot (Supplementary Fig. 1).
All lines were subject to quality control as previously described 41 and included veri cation of pluripotency by immuno uorescent staining for pluripotency markers (Nanog, Tra1-60, SSEA4 and OCT3/4), veri cation of normal karyotype and veri cation of normal pro le on genome stability test (Supplementary Fig. 1).

iPSC culture and dopaminergic neuron differentiation
All cell culture reagents used, and media compositions are depicted in Supplemental Table 5. Midbrain neuronal precursor cells (NPCs) and dopaminergic neurons were generated following previously established protocols 42,43 .Brie y, iPSCs were dissociated with Gentle Cell dissociation reagent and transferred to uncoated asks in NPC Induction Media to allow for embryoid bodies (EB's) to form.EBs were cultured for 7 days and then transferred to polyornithine/laminin coated asks and grown for another 7 days in NPC induction media.To expand NPCs the EBs were then dissociated into small colonies by trituration in Gentle Cell dissociation media and replated as a monolayer on polyornithine/laminin coated asks.After reaching con uence, NPCs were harvested and frozen in FBS with 10% DMSO and stored in liquid nitrogen.
To differentiate neurons, NPCs were thawed in NPC Maintenance Media with Y-27632 (ROCK inhibitor) and plated on polyornithine/laminin.NPCs were grown for 5-9 days until con uent.For nal differentiation into dopaminergic neurons, NPCs were dissociated using Accutase and plated on polyornithine/laminin in Dopaminergic Differentiation Media.After 5 days, media was supplemented with mitomycin C to remove proliferative cells.Dopaminergic neurons were maintained by exchanging 1/3 of the culture volume for fresh dopaminergic differentiation media every 5-7 days.Neurons from every batch were assessed by immuno uorescence for expression of Map2 and TH (Supplementary Fig. 2A,B), and only batches achieving at least 50% Map2/TH positivity after 4 weeks of differentiation were used for the experiments included in this manuscript.
For high-content imaging experiments neurons were plated on 96-well plates at a density of 15,000 cells per well.For protein and RNA isolation experiments neurons were plated on 6-well plates at a density of 750,000 cells per well.For live-imaging experiments, neurons were plated on 4-chamber imaging dishes at a density of 100,000 cells per well.

Organoid culture, treatment, and imaging
The patient derived iPSCs with SNCA triplication mutation (3xSNCA) and corresponding SNCA knockout (SNCA-KO) were previously described 44 and provided by Dr. Tilo Kunath.These cells were used to generate midbrain organoids following a protocol previously established in our labs 45,46 .Three months after organoid induction they were treated with either DMSO (vehicle) or 1 µM CA074me and treatment was maintained for 60 days.All organoids (12 per group) were xed, cryo-sectioned, and prepared for immuno uorescence using antibodies against Map2, TH, α-syn and pSyn-129 as previously described 45 .Veri cation of expression of TH and absence of α-syn in SNCA-KO in the organoids used in this study is depicted in Supplementary Fig. 3. Images were acquired using the Leica TCS SP8 confocal microscope and image analysis was performed with an in-house developed script for quanti cation of immuno uorescent signal in organoids (https://github.com/neuroeddu/OrgQ).
Lentivirus was used to stably transduce parental CRISPRa and CRISPRi cell lines which then underwent puromycin selection to generate polyclonal cell lines expressing the gRNA of interest.For each target, several gRNAs were tested and the best performing sequences were selected by assessing target modulation by RT-qPCR analysis of gene expression.

Drug and PFF treatments
CA074me (Selleckchem) and PADK (Bachem) treatments were performed at the indicated nal concentrations with DMSO as vehicle.For PFF experiments in Figs. 2 and 3, a single drug treatment was performed simultaneous with PFF administration, after which media was refreshed every 5-7 days.For lysosomal assays in Fig. 3, drug was administered 5 days prior to the assay unless otherwise speci ed.
For PFF treatments on RPE1 cells, 50,000 cells were plated on 12-well plates.After 24 hours, PFF was added and cells were allowed to continue growing for 48 hours before cells were washed with PBS and dissociated with trypsin to remove non-internalized PFFs before being lysed in RIPA buffer.
For PFF high-content imaging assays with PFF, neurons were treated with PFF after 2 weeks of differentiation in 96-well plates.After treatment, media was refreshed every 5-7 days normally.At completion of treatment, cells were washed with PBS and xed with 4% paraformaldehyde.

High-content imaging -immuno uorescence
Cells were permeabilized for 10 minutes with 0.3% saponin (lysosome immunostaining) or 0.2% triton X-100 (pS129-α-syn assay and TFEB assay) in PBS and blocked with 1% BSA, 4% goat-serum and 0.02% triton X-100 in PBS.Antibodies used are described in Supplemental Table 7. High content imaging was performed on an Opera Phenix high-content confocal microscope (Perkin Elmer) and image analysis was performed using Columbus (Perkin Elmer).Data processing was then conducted using R studio as previously described 52 .Brie y, nuclei were rst identi ed by the Hoechst channel, and surrounding soma was identi ed as Map2-positive region.Relevant secondary stains were then quanti ed within this Map2de ned region.Single-cell data were then processed using a custom script in R studio to lter objects based on nuclear size, nuclear shape and Map2 staining intensity to identify only the neuronal cells for inclusion in subsequent analysis.
High content imaging -live cell assays PFB-FDGlu GCase activity assay: Cells in 96-well plates were pre-loaded for 30 minutes with lysotracker deep red (1:20,000, Invitrogen).Media was then exchanged for FluoroBrite imaging media (Thermo) containing 25uM of PFB-FDGlu (Invitrogen) and cells were then imaged on the Opera Phenix every 15 minutes for 2 hours to monitor GCase activity.Using the Columbus software, lysotracker signal was used to identify cells for quanti cation of GCase substrate uorescence, which is depicted as the mean uorescence per cell.
DQ-BSA: Cells were pre-loaded with DQ-BSA (Invitrogen) for the indicated duration in standard culture media.Cells were then stained with lysotracker deep red (1:20,000, Invitrogen) for 30 minutes and media was exchanged for FluoroBrite and imaging was conducted on the Opera Phenix.Using the Columbus software, lysotracker signal was used to identify cells and DQ-BSA uorescence intensity was measured.

Whole-cell proteomics mass spectrometry
For proteomics experiments on iPSC-derived DA neurons, 750,000 neurons were plated on 6-well plates and differentiated for 3 weeks.After 3 weeks, neurons were treated with CA074me and/or 300 nM of PFF and then maintained normally for 3 weeks.No additional drug or PFF were added during the maintenance period.Sample processing, mass-spectrometry and data analysis was performed by The Proteomics and Molecular Analysis Platform at the Research Institute of the McGill University Health Centre (RI-MUHC).
Samples were processed for TMT labelling according to the manufacturer recommendations (ThermoFisher TMT-16plex reagents).Labelled peptides were fractionated using Pierce™ High pH Reversed-Phase Peptide Fractionation Kit into 8 fractions.Each fraction was re-solubilized in 0.1% aqueous formic acid and 2 micrograms of each was loaded onto a Thermo Acclaim Pepmap (Thermo, 75uM ID X 2cm C18 3uM beads) precolumn and then onto an Acclaim Pepmap Easyspray (Thermo, 75uM X 15cm with 2uM C18 beads) analytical column separation using a Dionex Ultimate 3000 uHPLC at 250 nl/min with a gradient of 2-35% organic (0.1% formic acid in acetonitrile) over three hours running the default settings for MS3-level SPS TMT quantitation 53 on an Orbitrap Fusion instrument (ThermoFisher Scienti c) operated in DDA-MS3 mode.
To translate .rawles into protein identi cations and TMT reporter ion intensities, Proteome Discoverer 2.2 (ThermoFisher Scienti c) was used with the built-in TMT Reporter ion quanti cation work ows.Default settings were applied, with Trypsin as enzyme speci city.Spectra were matched against the human protein fasta database obtained from Uniprot(2022).Dynamic modi cations were set as Oxidation (M), and Acetylation on protein N-termini.Cysteine carbamidomethyl was set as a static modi cation, together with the TMT tag on both peptide N-termini and K residues.All results were ltered to a 1% FDR.
Pathway analysis of differentially abundant proteins was conducted using STRING 54 .

Western Blot
Cultured cells were washed with PBS and collected in RIPA lysis buffer with protease inhibitors.Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scienti c™) and proteins were prepared at the desired concentration in 6X Laemmli buffer and heated at 95°C for 5 minutes.20 µg of protein were loaded on polyacrylamide gels, run with SDS running buffer and transferred onto nitrocellulose membranes and blocked for 30 mins in 5% skim milk made in 1X PBS with 0.1% Tween.
For alpha-synuclein blots membranes were xed using 4% PFA and 0.1% glutaraldehyde for 30 minutes before blocking.Blocked membranes were incubated with primary antibody (Supplemental Table 7) at 4°C overnight followed by HRP-conjugated secondary antibodies for 90 mins at room temperature.
Protein detection was performed by chemiluminescence using Clarity Western ECL Substrate (Biorad) and Western blots were quanti ed using ImageJ.

RNA Extraction and qRT-PCR
RNA isolation was performed using the RNeasy Mini Kit (Qiagen) and cDNA was generated by RT-PCR using the MMLV Reverse Transcriptase kit (Thermo) with random hexamer primers.Real-time quantitative PCR was performed using SSoAdvanced SYBR Green Master Mix (Biorad).Primers for speci ed target genes were designed using NCBI PrimerBlast (Supplemental Table 8).

Live cell confocal imaging and analysis
Neurons were plated on CELLview™ 4-chamber imaging dishes (Greiner) at 100k cells per well.After 3 weeks of differentiation neurons were treated with alexa633 labelled PFF and/or CA074me.After 72 hours neurons were washed and incubated with 50nM of Lysotracker™ Green DND-26 (Invitrogen) for 30m at 37°C in standard culture media.The dye solution was exchanged for FluoroBrite™ DMEM (Gibco) and plates were immediately imaged.Images were acquired on a custom Andor spinning disc confocal microscope at 100X magni cation.Single frames were acquired for cell bodies (488nM Lysotracker, 647 PFF).For neuronal tra cking movies, frames were acquired every 1. 5s for a total of 61 frames.
For the analysis of somatic lysotracker and PFF colocalization cell bodies were masked manually using FIJI ImageJ.For each image, Lysotracker and PFF signal were binarized using Otsu automatic thresholding, and binarized co-cluster signal was obtained using the "Image Calculation > > AND" function.Somatic slice densities of Lysotracker, PFF, or co-clusters were calculated via "Analyze Particles".Finally, percentage of PFF in lysosomes was obtained by normalizing co-cluster particle density to Lysotracker density, and percentage of lysosomes containing PFF was similarly obtained by normalizing co-cluster particle density to PFF particle density.
Lysosomal motility was analyzed using the FIJI ImageJ TrackMate plugin 55,56 .The generated tracks were then ltered by max track speed and then analyzed using Python.

Results
Variants in CTSB likely drive the association with PD and are associated with CTSB expression in multiple brain regions.
Variants in the genetic locus containing CTSB are signi cantly associated with risk of PD 9 yet this locus includes multiple other genes including FDFT1, NEIL2, GATA4 and it remains uncertain whether CTSB itself drives the association.We examined all the variants that are 1MB upstream or downstream to the top GWAS variant in this locus and using GCTA-COJO, we show that an intronic CTSB variant (rs1293298, p = 3.41E-16, located in intron 1 of CTSB within a potential enhancer region) is the top variant associated with PD risk, without secondary associations.Fine mapping using FINEMAP gave this variant the highest posterior probability (0.127) of being causal, of all nominated variants.This variant is in LD with multiple variants in this locus (r 2 > 0.8) that are associated with CTSB expression with H4 posterior probability > 0.8 in multiple brain regions.The associations between genetic variants, PD and CTSB expression in PDrelevant brain regions such as basal ganglia, cortex and nucleus accumbens are depicted in Fig. 1.In particular, the minor allele of the rs1293298 CTSB variant linked to PD in GWAS exhibits a protective effect against PD and is associated with elevated expression levels of CTSB in brain tissues relevant to the disease (Fig. 1A-D).Analysis using SMR and HEIDI suggests that the QTLs in CTSB are potentially causally linked to PD with p HEIDI > 0.05 in multiple tissues (i.e., we could not reject the null hypothesis that there is a single causal variant affecting both gene expression and risk for PD).All results from the GCTA-COLOC, FINEMAP, SMR and HEIDI analyses are detailed in Supplementary Tables 9-11.
These common CTSB variants occur in non-coding regions and likely exert their effects through altering expression levels.However, given the evidence that protective CTSB variants are associated with increased mRNA expression levels, we hypothesized that loss of function coding variants in CTSB would be likely to promote PD risk.We conducted rare variant analysis in 5,801 PD cases and 20,427 controls from six cohorts (Supplementary Table 2).We observed a nominal association between all rare variants and variants with high CADD score and PD risk in the Sheba cohort (p = 0.03 and p = 0.049, respectively).
However, upon examining other cohorts and conducting a meta-analysis we did not nd any additional associations (Supplemental Table 12).Additionally, we studied the role of rare CTSB variants on the age of PD onset.We found nominal association between functional variants and age at onset in McGill cohort (p = 0.044) and in the meta-analysis for functional and non-synonymous variants (p = 0.048 and p = 0.043, respectively).All these results should be interpreted with caution as no p-values survived multiple comparisons.

CatB inhibition promotes α-syn aggregation in dopaminergic neurons
To functionally interrogate the role of catB in the handling of α-syn brils we generated iPSC-derived dopaminergic neurons 41,42 and treated them with pre-formed α-syn brils (PFFs) and the catB inhibitor CA074me (1 µM) 58 .Exposure to PFFs promotes endogenous α-syn aggregation which can recapitulate many features of Lewy pathology including the accumulation of S129-phosphorylated α-syn (pSyn-S129) 59 .We used high-content confocal imaging to quantify pSyn-S129 in Map2-positive neurons following exposure to PFF and/or CA074me (Fig. 2A).After 2, 3 or 4 weeks, a single treatment with CA074me administered at the time of PFF exposure increased the abundance of pSyn-S129 (Fig. 2B).Similar We performed whole-cell proteomics with TMT labelling to characterize the broader impact of PFF or CA074me treatment on human DA neurons (Supplementary Table 13).We found that 3 weeks after treatment CA074me had minimal residual effects, while PFF exposure signi cantly altered the abundance of 60 proteins (Fig. 1C -Venn diagrams).GO-term analysis with STRING 54 revealed that the predominant pathways impacted by PFF treatment were the downregulation of proteins involved in cellular adhesion and cytoskeletal organization (Fig. 1C -bar graphs).Combining CA074me with PFF resulted in > 2x the number of differentially abundant proteins than either treatment alone.GO-term enrichment revealed many similar pathways were downregulated by either PFF or PFF + CA074me, but the combined treatment also upregulated pathways not found to be altered by PFF alone.

CatB inhibition induces lysosome dysfunction in dopaminergic neurons
Extracellular α-syn aggregates are taken into neurons by a variety of mechanisms and are rapidly tra cked to lysosomes 60,61 .To determine whether CA074me treatment altered the tra cking of PFFs into lysosomes or their persistence there, we performed live cell confocal imaging of DA neurons exposed to alexa-633 tagged PFFs (PFF-633) for 72 hours and stained with lysotracker (Fig. 3A).CA074me increased the overall lysosome content (Fig. 3B) and the density of PFF-633 uorescent puncta per cell (Fig. 3C) but colocalization of PFF-633 with lysosomes was unchanged (Fig. 3D).We interpret these observations as indicating that although the abundance of both lysosomes and PFF-633 within each cell is slightly elevated in CA074me treated neurons, the proportion of PFF-633 tra cked to lysosomes is unaffected.
Given the observed increase in lysotracker density, we next sought to further characterize how catB inhibition affected lysosome abundance and function in human DA neurons.Similar to lysotracker, the abundance of the lysosomal membrane protein LAMP1 was increased after CA074me treatment, independent of concurrent PFF exposure (Fig. 3E, F).However, the degradative capacity of lysosomes (measured using the uorogenic probe DQ-BSA) was reduced (Fig. 3G).The speed of lysosomal tra cking in neuritic projections was also reduced following CA074me (Fig. 3H).Lastly, given the genetic interaction between variants in CTSB and GBA1 in PD risk 10 and that catB has been found to regulate glucocerebrosidase (GCase) activity in HEK293 cells 62 we assessed the impact of catB inhibition on lysosomal GCase activity in DA neurons using the uorogenic probe PFB-FDGlu (Fig. 3I-K).CatB inhibition with either CA074me or PADK impaired lysosomal GCase activity (Fig. 3J, K).These observations indicate that despite increasing lysosome abundance, catB inhibition impairs several aspects of lysosome function in DA neurons, including degradative capacity, tra cking and GCase activity.
To determine whether altered lysosome function could be related to accumulation of α-syn aggregates, (which has been found to impact lysosomal hydrolase tra cking 11,13 ) we differentiated 3xSNCA and SNCA-KO iPSCs 45 and treated them with CA074me for 3 weeks.We observed that while total levels of αsyn were unchanged by CA074me (Fig. 3L), LAMP1 was increased in both 3xSNCA and SNCA-KO neurons (Fig. 3M) indicating the increase in lysosome content is independent of α-syn.We additionally stained with antibodies that preferentially detect aggregated species of α-syn (Syn303) and found this to be increased in CA074me-treated SNCA-triplication neurons (Fig. 3N), although almost no S129-pSyn signal above background was detected in these cells (Fig. 3O).

CTSB levels regulate PFF clearance in RPE1 cells
While CA074me is selective for catB, it has been reported to inhibit other cathepsins, albeit at concentrations greater than those used in this study 63,64 .RNA sequencing of our iPSC-derived DA neurons indicated that 12 cathepsin family members were expressed at the RNA level (data not shown), including CTSD and CTSL which were previously found to cleave α-syn in vitro 18-20 .To determine how individual cathepsin species contribute to brillar α-syn clearance in a cellular context we used CRISPRinterference (CRISPRi) to generate RPE1 cell lines in which CTSB, CTSD, CTSL or α-syn (SNCA) were stably repressed (denoted CTSBi, CTSDi, CTSLi and SYNi respectively) as well CRISPR-activation (CRISPRa) to upregulate CTSB (CTSBa) (Fig. 4A,B).Endogenous α-syn protein was undetectable in SNCAi cells and modestly elevated in CTSBi cells (Fig. 4C, D).In contrast, CTSBa had no effect on endogenous α-syn protein (Fig. 4E, F).Strikingly, 48 hours after exposure to PFFs, CTSBi cells (but not CTSDi or CTSLi) exhibited signi cantly greater accumulation of α-syn aggregates compared to control cells (Fig. 4G,H) while CTSBa had the opposite effect, modestly reducing the levels of aggregated α-syn (Fig. 4I, J).This effect was recapitulated by treatment of control or SNCAi cells with CA074me (Fig S5A ,B) indicating that this accumulation re ects either increased cellular uptake or failed clearance of the PFFs, rather than new aggregate seeding.
To determine whether the uptake or clearance of PFFs was affected, we used Alexa-633 uorescently labelled PFFs and conducted a pulse-chase experiment to monitor the uptake and subsequent clearance of PFFs (Fig. 4K, L).During a 3h exposure, or 3-hour exposure with short washout (3h chase), the PFF-633 levels per cell were similar across cell lines.However, when washout was extended to 21-hours CTSBi cells retained more PFF-633 (Fig. 4L), suggesting impaired clearance.CTSDi and CTSLi also appeared to impair PFF clearance in this assay but to a lesser extent than CTSBi.Similar to what we observed by Western blot, when we exposed CTSBa cells to PFF-633 for 48 hours, we observed a reduced accumulation of the tagged PFF (Fig. 4M).Taken together, these ndings indicate that CatB regulates the clearance of internalized α-syn aggregates in lysosomes.

CTSB repression impairs autophagy and lysosomal function in RPE1 cells
We next used our CTSBi cell line to further interrogate the role of CTSB in regulating lysosome abundance and overall lysosome function.Given the accumulation of lysosomal structures in DA neurons we hypothesized that loss of catB caused a state of lysosomal dysfunction that resulted in the accumulation of hypo-functional lysosomes.However, a previous study found that loss of catB also triggered lysosome biogenesis, and in select circumstances had a net effect of enhancing clearance of lysosomal cargo 65 .
CTSBi cells had signi cant accumulation of lysosomes, as indicated by an increase in LAMP1 and LAMP2 immuno uorescent signal (Fig. 5A-C), an increase in LAMP1 and GCase protein levels (Fig. 5D-F) and increased number and size of electron dense lysosome-like structures (including lysosomes and multivesicular bodies) observed by electron microscopy (Fig. 5G).However, despite having increased lysosome content and increased GCase protein levels, the activity of lysosomal GCase per cell was signi cantly reduced in CTSBi cells (Fig. 5H, I).
To determine whether impairment in autophagic ux could contribute to the accumulation of lysosomes, we measured the abundance of p62 puncta under fed and starved conditions, and in the presence of ba lomycin (to inhibit lysosomal clearance of autophagosomes).CTSBi resulted in increased abundance of p62 puncta under fed and serum-starved conditions, but not in the presence of ba lomycin (Fig. 5J, K) suggesting an impairment in the clearance of autophagosomes.Similarly, we observed accumulation of the autophagy-associated proteins LC3B and p62 by western blot in CTSBi cells (but not CTSDi or CTSLi), in the absence of serum starvation (Fig S6A ,B).

While this impaired autophagosome clearance likely contributes to the accumulation of lysosomes in
CTSBi cells, catB has previously been reported to regulate lysosome biogenesis via activation of the transcription factor TFEB 65,66 .Given that mRNA levels of CTSD and CTSL were already noted to be increased in CTSBi cells (Fig. 4A) we suspected a role for increased TFEB activity.Indeed, we found that in CTSBi cells, nuclear localization of TFEB was increased in non-starved cells (Fig. 5L, M) and mRNA levels of several lysosomal genes (including LAMP1, GBA and MCOLN1) were transcriptionally upregulated (Fig. 5N).These results taken together indicate that a combination of impaired lysosome turnover and upregulated lysosome biogenesis contribute to the increased abundance of dysfunctional lysosomes in RPE1 cells lacking CTSB.

Knockout of CTSB in human dopaminergic neurons leads to lysosomal dysfunction
To further con rm the lysosomal phenotypes that we previously observed in neurons treated with CA074me, we generated CTSB knockout iPSCs (CTSB-KO) and differentiated them into dopaminergic neurons (Fig. 6A, B).Similar to CA074me treatment, in CTSB-KO neurons lysosome abundance measured either by LAMP1 immuno uorescence (Fig. 6C) or lysotracker (Fig. 6D) were increased, while degradative capacity (Fig. 6E) and neuritic tra cking velocity (Fig. 6F) were reduced.Lysosomal GCase activity was likewise reduced by approximately 20% in CTSB-KO neurons (Fig. 6G, H) although GCase protein levels were unaffected (Fig. 6I, J).Unlike CTSB-knockdown RPE1 cells, CTSB-KO neurons did not exhibit detectable activation of TFEB (Fig S7A) or transcriptional upregulation of lysosomal genes (Fig S7B) arguing that catB may regulate TFEB activity and lysosome biogenesis in a cell-type or context-dependent manner.
CTSB de ciency promotes synuclein pathology in human dopaminergic neurons and midbrain organoids CTSB-KO DA neurons were found to have modestly elevated levels of endogenous α-syn (Fig. 7A,B).
When treated with PFFs for 72 hours CTSB-KO neurons did not exhibit higher levels of total α-syn (Fig. 7A,   C).However, 3 and 4 weeks after PFF treatment CTSB-KO neurons accumulated signi cantly more pSyn-S129, and this was evident when measuring either the average pSyn-S129 intensity within Map2-positive neurons, or the percentage of pSyn-positive cell bodies (Fig. 7D-F).The e ciency of PFF uptake (measured by alexa-488 tagged PFF internalization) was unaffected in CTSB-KO neurons (Fig. 7G,H To determine whether the loss of catB function could promote α-syn aggregation independent of PFF exposure we generated midbrain organoids from patient-derived iPSCs harboring an SNCA triplication mutation (Fig S3).We have previously found that these organoids spontaneously develop pSyn-S129positive α-syn aggregates after sustained culture 45 .We treated 3x-SNCA or isogenic SNCA-KO organoids for 60 days with DMSO (vehicle) or 1 µM CA074me and observed an increase in the abundance of pSyn-S129 (measured as the area positive for pSyn-S129 relative to total organoid area) (Fig. 8A-C), while treatment had no effect on total neuron content (Map2-positive area) (Fig. 8D).These ndings further indicate that catB contributes to lysosomal clearance of both endogenous α-syn and exogenously applied α-syn PFFs.

Discussion
Cathepsin B has previously been suggested to contribute to the degradation of α-syn and genetic variants in the CTSB locus are signi cantly associated with PD, suggesting that this lysosomal protease may play an important role in the disease.Here we provide genetic and functional evidence supporting the crucial involvement of CTSB in PD, speci cally relating to the function of lysosomes and degradation of α-syn aggregates in dopaminergic neurons.
Firstly, our genetic analysis provides compelling evidence for a causal relationship between common noncoding variants in the CTSB gene and both brain expression levels of CTSB and PD risk.This genetic analysis indicates that of the variants and genes in the CTSB GWAS locus, the association is most likely driven by CTSB variants that affect its expression in different brain regions.In particular, the minor allele of the rs1293298 CTSB variant, linked to PD in GWAS, exhibits a protective effect against PD and is associated with elevated expression levels of CTSB in several brain tissues.This nding is also supported by recent work in which we have used machine learning to nominate the most likely causative genes in each known PD locus, in which CTSB was also found to be the top nominated gene 67 .
One potential mechanism by which CTSB variants may in uence PD risk is through the ability of catB to cleave and degrade α-syn 18-20 .However, while several cathepsins appear capable of cleaving α-syn in vitro, CTSB alone stand out as a genetic risk factor for PD.By using genetic tools to modulate the expression levels of CTSB, CTSD and CTSL in RPE1 cells, we show that in a cellular context, CTSB is particularly critical for the maintenance of lysosome function and clearance of brillar α-syn.This is supported by recent the nding that while many cathepsins exhibit redundancy, the sites within α-syn cleaved by CTSB are relatively unique, and unlikely to be compensated for by other cathepsins 20 .This lack of redundancy may explain why CTSB stands out as a genetic risk factor and an essential mediator of α-syn clearance.
In addition to a potential direct role of catB in degrading α-syn aggregates, we have also observed that catB impairment leads to lysosome accumulation and broad impairment of lysosome functions, including impaired GCase activity.Variants in CTSB and GBA interact to mediate genetic risk for PD 10 and given the established importance of GCase in mediating risk of synucleinopathy (reviewed in 68 ) this raises the question of whether the impaired α-syn clearance observed following catB impairment is partially mediated by secondary GCase impairment.This loss of GCase activity occurs despite an increase in overall lysosome content, and in the case of RPE1 cells, an increase in GCase protein levels.
One potential mechanism linking catB to GCase activity is via the ability of catB to cleave pro-saposin into saposin C which acts as a co-activator of GCase 62 .Future studies will be required to determine the importance of GCase as a mediator of catB -dependent α-syn clearance, as well as the mechanism of interaction between these lysosomal proteins.
In the present work we have demonstrated using several cellular models that loss of CTSB impairs GCase activity and promotes the accumulation or aggregation of α-syn after exposure to preformed α-syn brils.
These ndings complement genetic evidence that CTSB variants associated with increased expression levels are protective against the disease and provides potential mechanistic support for the genetic interaction between CTSB and GBA.Beyond the direct genetic association, impaired catB expression or activity have also been reported in cellular or animal models associated with PD-risk factors like αsyn/SNCA, GBA, TMEM175 and LRRK2 [11][12][13][14][15][16][17]69 . Togther this evidence highlights CTSB as an important player in the etiology of synucleinopathies such as Parkinson's disease, and further study of its biology may help to uncover novel therapeutic approaches to this disease.
) but as expected LAMP1 was elevated (Fig. 7G,I).Using live-cell confocal microscopy, we observed a modest increase in total PFF-633 levels in CTSB-KO neurons 72 hours after treatment (Fig S8A, B), however there was no difference in the tra cking of alexa-633 tagged PFFs to lysosomes as measured by PFF-633 and lysotracker colocalization (Fig S8C).

Figures Figure 1
Figures

Figure 7 CTSB
Figure 7 57M) coupled to a camera (Gatan Ultrascan 4000 4 k × 4 k CCD Camera model 895).The identi cation of cellular elements was based on standard descriptions57.Statistical AnalysisStatistical analysis was conducted in GraphPad Prism9 software.For experiments with iPSC-derived neurons biological replicates were de ned as experiments conducted at different times from the same batch of banked NPCs, or as experiments conducted in parallel from different batches of NPCs.A minimum of 3 distinct batches of NPCs were used for each experiment.Statistical comparisons were performed using t-tests (only 2 conditions), Bonferroni-corrected t-tests (more than 2 conditions compared).Signi cance levels are depicted in gure legends.
aqueous osmium tetroxide (Mecalab) for 1 h at 4°C, and stained with 4% uranyl acetate (EMS) in 70% ethanol for 45min at 4°C.After dehydrations in ascending alcohols, cells were embedded in Epon resin (Mecalab), and cut at 75 nm thickness in the ultra microtome.Sections were collected in 200 Mesh cooper grids (EMS) and stained with 4% uranyl acetate for 5min following by Reynold's lead citrate for 2 min.Sections were visualized using a transmission electron microscope (Tecnai G2 Spirit Twin 120 kV Cryo-