ML198 | Inhibitor Kits

N370S-GBA1 Mutation Causes Lysosomal Cholesterol Accumulation in Parkinson’s Disease

Patricia Garcı´a-Sanz, PhD,1,2 Lorena Orgaz, MSc,1,2 Guillermo Bueno-Gil, MSc,1,2 Isabel Espadas, PhD,1,2 Eva Rodrı´guez-Traver, MSc,1,2 Jaime Kulisevsky, MD, PhD,2,3 Antonia Gutierrez, PhD,2,4 Jose´ C. Da´vila, PhD,2,4 Rosa A. Gonza´lez-Polo, PhD,2,5 Jose´ M. Fuentes, PhD,2,5 Pablo Mir, MD, PhD ,2,6 Carlos Vicario, PhD,1,2† and
Rosario Moratalla, PhD1,2†*
1Instituto Cajal, CSIC, Madrid, Spain
2CIBERNED, Madrid, Spain
3Movement Disorders Unit, Neurology Dpt, Hospital Sant Pau (IIB-Sant Pau), Univ. Auto`noma de Barcelona, Barcelona, Spain
4Dpto. de Biolog´ıa Celular, Gene´tica y Fisiolog´ıa, Facultad de Ciencias, IBIMA, Universidad de Ma´laga, Ma´laga, Spain
5Dpto. de Bioqu´ımica, Biolog´ıa Molecular y Gene´tica F. Enfermer´ıa y T.O., Univ. de Extremadura, Ca´ceres, Spain
6Neurology Dpt, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Roc´ıo/CSIC/Universidad de Sevilla, Sevilla, Spain

ABSTRACT:

Background: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme b-glucocerebrosidase-1, increase the risk of developing Parkinson’s disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient- specific cellular model of PD.
Methods: We isolated fibroblasts from 4 PD patients carry- ing the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic retic- ulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry.
Results: The N370S mutation produced a significant reduction in b-glucocerebrosidase-1 protein and enzyme activity and b-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded pro- tein response and Golgi apparatus fragmentation. Fur- thermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autoph- agy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death.
Conclusions: Our results support a connection between the loss of b-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD patho- genesis, providing evidence that GBA1-PD shares com- mon features with lipid-storage diseases. VC 2017 International Parkinson and Movement Disorder Society

Key Words: multilamellar bodies; autophagic vesicles; mitochondria; ER stress; cell death

*Correspondence to: Prof. Rosario Moratalla, Cajal Institute, CSIC, Av. Dr. Arce 37, 28002 Madrid, Spain; [email protected]
Rosario Moratalla and Carlos Vicario contributed equally to this article.
Relevant conflicts of interests/financial disclosures: Nothing to report.
Funding agencies: This work was supported by grants from the Spanish Ministeries of Econom´ıa y Competitividad, Sanidad Pol´ıtica Social e Igualdad and ISCIII CIBERNED: SAF2016-78207-R, PCIN2015-098, CB06/05/0055 and PI2015-2/02 (to R.M.); SAF2013-4759R and CB06/05/0065 (to C.V.);
CB06/05/004 and PI15/00034 and Junta de Extremadura: GR15045 (to J.M.F.); FIS PI14/00170 to RA G-P; FIS PI15/00796 (to A.G.); La Caixa and
Marato´ TV3 (to J.K.). J.K. has received honoraria for lectures and/or advisory boards from AbbVie, Zambon, UCB, Italfarmaco, and TEVA. E.R.T., L.O., I.E., and G.B.G. were supported by FPI and FPU fellowships from MINECO and MECD. RA G-P was supported by the Contract for the Retention and
Attraction of Talent Researcher of the Government of Extremadura, TA13009 (Junta de Extremadura, Spain). P.M. has been supported by grants from the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (PI16/01575), the Consejer´ıa de Econom´ıa, Innovacio´n, Ciencia y Empleo de la Junta de Andalucı´a (CVI-02526, CTS-7685), the Consejer´ıa de Salud y Bienestar Social de la Junta de Andaluc´ıa (PI-0471/2013), the Socie- dad Andaluza de Neurolog´ıa, the Fundacio´n Alicia Koplowitz, and the Fundacio´n Mutua Madrilen~a.
Received: 14 March 2017; Revised: 29 June 2017; Accepted: 29 June 2017
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.27119
VC 2017 International Parkinson and Movement Disorder Society

Introduction:
Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by dopa- minergic neuron loss in the substantia nigra pars com- pacta and the presence of Lewy bodies containing a-synuclein (a-syn) and ubiquitin.1 Heterozygous mutations in the GBA1 gene encoding the lysosomal hydrolase b-glucocerebrosidase-1 (GCase1), involved in the breakdown of glucosylceramide (GlcCer), repre- sent the greatest genetic risk factor for PD and PD- associated Lewy body dementia.2,3 Homozygous GBA1 mutations result in Gaucher disease (GD), which is the most prevalent lysosomal storage disorder (LSD) characterized by decreased GCase1 activity and subsequent accumulation of GlcCer in several tissues, including the brain. However, the relation between decreased GCase1 activity and PD etiology is not yet fully understood. Both toxic gain of and loss of GCase1 function have been postulated,4 although such hypotheses are not mutually exclusive. The majority of GBA1 mutations do not directly affect the catalytic site; they instead destabilize its native structure, lead- ing to misfolded protein accumulation. This triggers an endoplasmic reticulum (ER) protein-folding quality control mechanism,5 resulting in ER stress upregula- tion and autophagy-lysosome dysfunction.6,7 Disrup- tion of GCase1 activity can increase GlcCer level (and that of related lipid derivatives) in lysosomes, which, in turn, promote a-syn aggregation and subsequent GCase1 activity inhibition.4,8,9 Although studies10-13 have shed light on the pathogenicity of GBA1 muta- tions, the molecular mechanisms underlying cellular homeostasis dysregulation in PD remain unclear.
Autophagy impairment impacts multiple pathways relevant to cellular homeostasis, leading to neurode- generative diseases.14 Autophagy is a bulk lysosomal degradation process for proteins, intracytoplasmic aggregates, lipids, and damaged organelles, including mitochondria.15 The lysosome is involved in later steps of autophagy because it fuses with the autopha- gosome to digest its content. Maturation of late endo- somes to lysosomes requires lipid metabolism.16 Indeed, cholesterol plays a key role in regulating lyso- somal membrane integrity and cell death,17 and its accumulation in lipid rafts has been found in several LSDs.18 In particular, Niemann-Pick disease type C (NPC) is characterized by excessive cholesterol storage in late endosomes/lysosomes,19 which causes lyso- somal dysfunction and disrupts GCase1 localization, leading to decreased GCase1 activity. Moreover, increased intracellular cholesterol modifies GCase1 processing in GD.20
To understand the role of GCase1 in cellular homeo- stasis, we studied the impact of N370S GBA1 mutation in fibroblasts from PD patients. We demonstrate a link between loss of GCase1 function, lysosomal cholesterol accumulation, and GBA1-PD pathogenesis.

Methods

Patients and Fibroblasts
We generated fibroblasts from skin biopsies of 4 PD patients with heterozygous mutation in the GBA1 gene (N370S/wild type), 6 healthy controls, 2 idio- pathic PD patients, and 1 type I-GD patient (L444P/ L444P), matched by age (Supplementary Table 1). We obtained informed consent from all the participants. To confirm this mutation in fibroblasts, we extracted DNA (IllustragenomicPrep Mini spin kit, GE Health- care Europe, Barcelona, Spain) and sequenced it at the CeGen (Santiago de Compostela, Spain) using Seque- nom technology. We conducted the experiments in all fibroblasts (1.2-1.5 3 104 cells/cm2) in parallel at low passage.

Western Blotting
We performed Western blotting (WB) from fibro- blast lysates as described previously.21 Primary anti- bodies for GCase1, cathepsin B, cathepsin D, LC3B, b-actin, b-tubulin (Cat. Nos. G4171, C6243, C0715, L7543, A5441, T4026; Sigma-Aldrich, St. Louis, MO, USA) Beclin1, CHOP, calnexin (Cat. Nos. sc-11427, sc-575, sc-6465; Santa Cruz, CA, USA), p62/SQSTM1 (Cat. No. H00008878-M01; Abnova), LAMP1 (Cat. No. GTX19294; Gentex, Irvine, CA, USA), SOD1 (Cat. No. 200-4191S; Rockland Immunochemicals, Philadelphia, PA, USA), and p-mTOR (Cat. No. 2971s; Cell Signaling Technology, Danvers, MA, USA) were used followed by appropriate secondary horse- radish peroxidase or infrared dye (LI-COR Biosci- ences, Lincoln, NE, USA)-conjugated antibodies, detected by chemiluminescence using ECL Substrate (BioRad, Hercules, CA, USA) or Odyssey Infrared Imaging System (LI-COR Biosciences), respectively. We used b-actin or b-tubulin as a loading control. For each study, at least 3 independent experiments were performed.

Endoglycosidase-H Treatment
Protein lysates were performed according to the manufacturer’s instructions (New England Biolabs, Hitchin, UK).

Immunocytochemistry
Fibroblasts were fixed with 4% paraformaldehyde (PFA) or methanol and incubated with the following primary antibodies: GCase1 (Cat. No. G4171; Sigma), GCase1, LAMP1 (Cat. Nos. sc-166407, sc-18821; Santa Cruz), p62/SQSTM1 (Cat. No. GP62-C; Progen, Heidelberg, Germany), GFP (Cat. No. 04404-84; Nacalai Tesque, Kyoto, Japan), and Cleaved Caspase-3 (Cat. No. 9661; Cell Signalling). The fibroblasts were then exposed to secondary antibody conjugated to Alexa Fluor 594 or 488. The nuclei were labeled with 1 lg/mL DAPI (ThermoFisher Scientific/Molecu- lar Probes, Waltham, MA, USA). Finally, the cells were mounted with Prolong Gold (ThermoFisher Sci- entific) and examined under an AF 6500-7000 fluores- cence or SP5 laser confocal microscope (Leica, Bensheim, Germany).

Cell Staining
We analyzed lysosomal mass using LysoTracker Red (ThermoFisher Scientific/Molecular Probes). The fibro- blasts were incubated with 70 nM LysoTracker (30 minutes), fixed in 4% PFA, rinsed with glycine, and then stained with 25 lg/mL Filipin (Sigma) for free cholesterol detection. To stably label autophagosomes, we infected fibroblasts with lentiviral particles22 encoding LC3B-GFP, provided by Dr. Shirihai (Boston University, USA). We collected GFP-positive cells by fluorescence-activated cell sorting and seeded them. The fibroblasts were fixed, DAPI-labeled, and mounted for image acquisition using a Leica fluores- cence microscope. The cells were grown on l-Slide 8- Well chambers (Ibidi Martinsried, Germany), loaded with a CytoPainter Golgi/ER Staining Kit (Abcam, Cambridge, MA, USA), and then analyzed using a Leica SP5 inverted confocal microscope.

Fluorescence Image Analyses
We conducted fluorescence analyses of LysoTracker, Filipin, and LC3B-GFP using NIH ImageJ software to calculate the integrated intensity (IntDen., signal inten- sity contained in the stained area within the cell). To quantify Filipin and LysoTracker-positive cells, images were acquired by a fluorescence microscope and ana- lyzed using Leica confocal software, and a colocaliza- tion plug-in was used to calculate Pearson correlation coefficient (PCC). These analyses were performed inside a selected region of interest per image, represen- tative of the cell. To quantify Filipin in lysosomes marked with LysoTracker-positive organelles, we superimposed a LysoTracker mask over Filipin images to determine Filipin integrated intensity as previously done.23 Double-immunofluorescence images were ana- lyzed similarly with PCC or with Mander’s overlap coefficient (MOC).24 The proportion of cells display- ing perinuclear lysosome clustering was determined as described previously.25 Cleaved Caspase-3 immunostaining was scored using NIH ImageJ software by counting positive cells from 5 fields per coverslip.

Lysosomal Enzymatic Activities
We measured GCase1 and -2, b-galactosidase, and b-hexosaminidase A activity by fluorimetry13 and cathepsin B activity using Magic Red cathepsin B

Apoptosis and Reactive Oxygen Species Measurements
To evaluate apoptosis, we used an Annexin-V-FITC/ PI Kit (Inmunostep, Salamanca, Spain).27 We used DHE and DiOC6(3) (ThermoFisher Scientific/Molecular Probes) to detect superoxide anion production and changes in the mitochondrial transmembrane potential (Dwm).28

Electron Microscopy
We performed electron microscopy (EM) on fibro- blasts fixed with 2.5% glutaraldehyde/4% PFA.29 We identified multilamellar bodies (MLBs), autophagic vacuoles (AVs), swollen mitochondria, and engrossed ER by visual inspection of EM micrographs using established criteria.29-33 To quantify MLBs, AVs, and swollen mitochondria in micrographs, we used ImageJ software (as in reference 29). Briefly, 50 photos from different areas of a minimum of 6 cells per experimen- tal individual were recorded at 20,0003 magnification and analyzed. Results were expressed as number of structures per cell.

Quantitative RT-PCR
We extracted RNA using an Illustra RNAspin kit (GE Healthcare, Buckinghamshire, UK) and retrotran- scribed it to cDNA. We performed quantitative PCR (qPCR) with SYBR Green detection (Applied Biosys- tems, Alcobendas, Spain) and the primers for GAPDH, GBA1, PERK, ATF6, CHOP, BiP, SOD1, and CAT (forward: 50-GTCGGAGTCAACGG ATT- 30, 50-CCAAGCCTTTGAGTAGGGTAAG-30, 50- GAGCTGTCGGACCTCGCAGTG-30, 50-TGCTTCCA GCAGCACCCAAGACTC-30, 50-CCAAGGGAGAAC CAGGAAACG-30, 50-GCCTGTATTTCTAGACCTGC C-30, 50-GCACTAGCAGCATGTTGAGC-30, and 50-T TTCCCAGGAAGATCCTGAC-30; reverse: 50-AAGCT TCCCGTTCTCAG-30, 50-CCCGTGTGATTAGCCTG- GAT-30, 50-GGCAGCTTCCTGTTCTTCCACATCTG- 30, 50-CCCAGCAACAGCAAGGACTGGC-30, 50-TCA CCATTCGGTCAATCAGAGC-30, 50-TTCATCTTGC CAGCCAGTTG-30, 50-GCGTTGATGTGAGGTTCCA G-30, and 50-ACCTTGGTGAGATCGAATGG-30). We performed qPCR in triplicate, and values were nor- malized to GAPDH mRNA levels using the double delta Ct method.34

Statistical Analysis
All data were obtained from at least 3 independent experiments and were normalized to control values where appropriate. For cell imagine studies, at least 100 cells representative of each experimental condition were analyzed. We used GraphPad Prism 6.0 to

FIG. 1. Reduction of N370S GCase1 level and activity along with its retention within the ER. A representative WB of GCase1 (A) and a histogram of GCase1 protein levels (B), which were significantly reduced in the mutants. (C) GCase1 enzyme activity (nmol h/mg) was significantly decreased, and GBA1 mRNA (D) was not reduced in the mutants. (E, E0, F) Immunofluorescence colocalization analysis of GCase1 (red) and the lysosomal marker LAMP1 (green) in N370S-PD fibroblasts. Insets of merged panels in E are enlarged in the adjacent images, and the colocalization mask is shown in the rightmost images. (E0) Intensity profile along the diagonal dashed line of the enlarged images. Note that colocalization was reduced in N370S-PD and almost absent in GD cells. (F) Quantification of GCase1 and LAMP1 colocalization using PCC. (G, H) GCase1 Endo-H-sensitive (low MW bands representing the GCase1 retained in the ER, arrowhead) and GCase1 Endo-H-resistant (high MW bands representing lysosomal mature GCase1, arrow) and their quantification (H). (I, J) Lactacystine, a proteasome inhibitor (Lact., 10 lm-24 hours) does not influence GCase1 level. Samples in the blots were processed in parallel. Data represent the mean 6 SEM of at least 3 independent experiments from all cell lines. **P < 0.01; ***P < 0.001 versus controls; ###P < 0.001 versus untreated; Student t test (B, J), Mann-Whitney U test (C, D, F), and 2-way ANOVA followed by Bonferroni post hoc analysis (J). Scale bar: 10 lm.nperform statistics. We used the Student t, Mann- Whitney U, and v2 tests or 2-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test, as appropriate. Statistical significance was set at P < 0.05.

Results

Reduction of N370S GCase1 Level and Activity Along With Its Retention Within the ER
The N370S mutation significantly diminished GCase1 protein levels and activity (Fig. 1A,B,C) with- out altering GBA1 mRNA (Fig. 1D) in N370S-PD fibroblasts. Interestingly, idiopathic PD (ID-PD) fibro- blasts did not show any of these alterations (Suppl. Fig. 1A,B,C,D). Because this reduced GCase1 was not a result of transcription failure, we determined whether this mutation affected the GCase1 maturation process and its levels in lysosomes (its final localiza- tion). In control fibroblasts, GCase1 accumulated in cytoplasmic punctate, colocalizing with LAMP1 (lyso- somal marker; Fig. 1E) but not with calnexin (Suppl. Fig. 2A), indicating that GCase1 was appropriately processed. In contrast, in GBA1-GD fibroblasts, GCase1 was scattered and colocalized with calnexin (Suppl. Fig. 2A), but barely with LAMP1 (Fig. 1E), suggesting that most of the homozygous mutant pro- tein was retained in the ER. In heterozygous N370S- PD fibroblasts, a portion of the N370S GCase1 was found in the puncta, devoid of LAMP1 (Fig. 1E), as also confirmed by the fluorescence intensity profiles (Fig. 1E0). Accordingly, N370S-PD fibroblasts showed intermediate levels of colocalization with calnexin (Suppl. Fig. 2A,A0). We quantified the degree of coloc- alization of GCase1 with LAMP1 or calnexin using the PCC or MOC, respectively. As shown in Figure 1F and Supplementary Figure 2B, this mutation signifi- cantly decreased PCC and increased MOC.
To further confirm maturation defects in N370S GCase1, we explored its intracellular traffic by WB of the glycosylated GCase1 after Endo-H digestion. Retained GCase1 in the ER carries N-linked glycans, which is sensitive to Endo-H cleavage, producing a low-molecular-weight (MW) band of GCase1 on WB (Endo-H sensitive). This band was identified by diges- tion with PNGase F (used as a band guide). In control fibroblasts, most GCase1 was Endo-H resistant, indi- cating that GCase1 reached the lysosomes (Fig. 1G,H). In contrast, in N370S, Endo-H-resistant GCase1 was significantly reduced (Fig. 1G,H), sugges- ting that GCase1 was retained in the ER and did not reach the lysosomes because of maturation failure. Lactacystin (Calbiochem, USA), a proteasome inhibi- tor, did not affect GCase1 from control or N370S fibroblasts (Fig. 1I,J), ruling out a contribution of pro- teasome degradation to GCase1 levels.

ER Stress and Activation of the Unfolded Protein Response in N370S-PD Fibroblasts
N370S GCase1 retention in the ER can activate ER stress and the unfolded protein response (UPR). We found a significant increase in mRNA expression (qRT-PCR) for PERK, CHOP, BIP, and ATF6 (UPR markers35; Fig. 2A). Supporting these findings, CHOP and calnexin (ER stress marker) were significantly upregulated (Fig. 2B,C,D). Notably, calnexin immu- nostaining was diffuse in N370S-PD fibroblasts, sug- gesting ER disorganization similar to that observed in GD, and control fibroblasts exhibited typical tubules in the peripheral ER (Fig. 2E, arrowheads).
Electron microscopy studies have revealed a normal ER structure in control fibroblasts, with cisternae of rough ER regularly organized in stacks (Fig. 2F). Cis- ternae lumens were narrow, showing many ribosomes (arrowheads) on the external face. In contrast, ER was dilated and disorganized in N370S fibroblasts (Fig. 2F). Moreover, we found a significant decrease in GCase2 (ER enzyme) activity in N370S fibroblasts because of this ER disruption (Fig. 2G). These findings suggest that N370S mutation triggers ER stress and UPR activation, leading to alterations in ER structure.

Golgi apparatus Morphological Alterations in N370S-PD Fibroblasts
We analyzed the ER and Golgi apparatus (GA) using CytoPainter Golgi/ER Staining because mis- folded GCase1 accumulation may result in morpho- logical alterations in both organelles.11 Confocal images revealed normal ER structure (Fig. 2H; tubules fused to each other), consistent with calnexin labeling (Fig. 2E). However, N370S fibroblasts had their ER homogeneously distributed over the cytoplasm, most densely in the perinuclear area. The GA displayed typ- ical semilunar shapes in controls (Fig. 2H), and in mutant fibroblasts it appeared disrupted or fragmented into dispersed smaller elements (Fig. 2H). Quantitative analysis revealed a higher proportion of fragmented GA cells in N370S-PD fibroblasts (Fig. 2I). Electron microscopy analysis showed typical stacks of cisternae forming a ribbon of GA in controls; in N370S fibro- blasts, these cisternae were shorter and formed minis- tacks (Fig. 2J). These findings indicate that N370S mutation alters GA structure.

Impaired Autophagy in N370S-PD Fibroblasts
Beclin1, which induces autophagosome formation and is localized and regulated at the ER membrane,36 was increased in N370S fibroblasts (Fig. 3A,B), indi- cating that N370S mutation could induce autophagy by ER stress. In basal conditions, mTOR phosphoryla- tion in N370S fibroblasts was significantly decreased (Fig. 3C,D), confirming that autophagy was induced.
FIG. 2. N370S-GBA1 mutation activates ER stress and UPR and causes GA fragmentation. (A) mRNA levels of ER stress and UPR marker genes (PERK, CHOP, BIP, and ATF6) were significantly increased in N370S fibroblasts. Similarly, CHOP and calnexin were increased in N370S fibroblasts (B, C, D). (E) In control fibroblasts, calnexin (an ER marker) is expressed in punctate and tubular-like staining (arrowheads). This pattern was disrupted in GD and N370S-PD fibroblasts where signal was more spread out. (F) EM images showing altered ER morphology in N370S-PD fibroblasts including ER cisternae expansion (traced in purple). Arrows indicate ribosomes. (G) GCase2 enzyme activity (nmol h/mg) was significantly reduced in mutant fibro- blasts. (H) Confocal images of N370S-PD and control fibroblasts stained with CytoPainter Golgi/ER Staining Kit illustrating the GA, green; ER, red; nucleus, blue, Nu. Note significant GA fragmentation in N370S-PD fibroblasts, in contrast to the semilunar shape seen in control cells. (I) Quantification of fragmented GA cells. (J) Electron micrographs illustrating that the GA of N370S cells contains fewer cisternae per stack and more vesicles than do control cells. Samples in the blots were processed in parallel. Data represent the mean 6 SEM of at least 3 independent experiments from all cells. *P < 0.05; **P < 0.01; ***P < 0.001 versus controls, Student t test (A, D, G, H) and v2 test (I). Scale bar: 10 lm, E, H; 2 lm, F; 0.5 lm, J.
FIG. 3. N370S mutation alters autophagic markers. Representative WBs and quantitative densitometry of Beclin1 (A, B) and p-mTOR (C, D). Repre- sentative LC3B-GFP images (E) and quantitative analysis (F) of fibroblasts treated with EBSS, rapamycin (Rap., 500 nM) or bafilomycin-A1 (Baf-A1, 100 nM) for 4 hours. Representative WB (G, I) and quantitative densitometry (H, J) of LC3II in fibroblasts after Baf-A1 (100 nM) for 4 hours and p62/ SQSTM1 after Baf-A1 (100 nM) plus EBSS for 4 hours. (K) Colocalization analysis of p62/SQSTM1 (red) and LC3B-GFP (green) in N370S-PD fibro- blasts under basal conditions and after NH4Cl (20 mM) and leupetine (Leup., 100 lM) plus EBSS treatment for 4 hours (L). Insets in the merged pan- els are enlarged in the adjacent images. Samples in the blots were processed in parallel; the data represent the mean 6 SEM of at least 3 independent experiments from all cell lines, *P < 0.05; **P < 0.01; ***P < 0.001 versus controls; #P < 0.05; ##P < 0.01; ###P < 0.001 versus untreated fibroblasts, Student t test (D) and 2-way ANOVA followed by Bonferroni post hoc analysis (B, F, H, J). Scale bar: 10 lm, E, K, L.As expected, Earle’s Balanced Salt Solution (EBSS)- induced starvation decreased p-mTOR in all fibroblasts. Next, we monitored autophagosome formation with LC3 and the cargo degradation with p62/SQSTM1. Rapamycin significantly potentiated the increase of LC3B-GFP-positive vesicles in mutant fibroblasts (Fig. 3E,F). Likewise, Baf-A1, which blocks autophagolysosome formation, significantly increased autophagosome accumulation in N370S fibroblasts (Fig. 3E,F). This result was confirmed by WB after Baf-A1 treatment (Fig. 3G,H), suggesting exacerbation of autophagosome synthesis in N370S cells. Alterna- tively, autophagosome accumulation could be also result of inhibition of lysosomal degradation, as shown by p62/SQSTM1 increases in mutant fibroblasts (Fig. 3I,J,K). However, Baf-A1 plus EBSS and leupeptin plus NH4Cl did not further potentiate p62/SQSTM1 accumulation (Fig. 3I,J,L). Autophago- some accumulation by Baf-A1 in the absence of changes in p62/SQSTM1 suggests that autophagy is impaired but not completely blocked in mutant fibroblasts.

N370S-GBA1 Dysfunctional Lysosomes
To determine if p62/SQSTM1 accumulation was a result of a degradation failure, we analyzed the autophagy-lysosome pathway. We found a significant increase in lysosomal mass detected by LysoTracker IntDen. (Fig. 4A,B) that was significantly enhanced by EBSS or rapamycin. This potentiation was confirmed by LAMP1 WB, and Baf-A1 treatment did not enhance the increase beyond that observed in the con- trols (Fig. 4C,D). Lysosomal mass was not altered in idiopathic-PD fibroblast (Suppl. Fig. 2C,D).
We examined lysosomal distribution in the cyto- plasm because it plays an important role in lysosomal function.37 Lysosomes were dispersed over the cyto- plasm in controls, and they clustered in the perinu- clear region in N370S-PD fibroblasts (Fig. 4E,F), suggesting lysosomal failure. This dysfunction was confirmed by significant b-galactosidase inhibition in N370S fibroblasts, although b-hexosaminidase-A remained unchanged (Fig. 4G). Moreover, N370S mutation significantly increased cathepsin B and D lev- els (Fig. 4H,I,J), although cathepsin B activity was decreased (Fig. 4K,L), indicating that N370S-GBA1 mutation alters lysosomal mass and function.

Cholesterol Accumulates in N370S Lysosomes
To determine whether decreased GCase1 activity had an effect on lipid metabolism, we examined free cholesterol levels using Filipin staining. In N370S cells, but not in idiopathic-PD, Filipin IntDen. was increased (Fig. 4M,P,Q and Suppl. Fig. 2C,D) and accumulated in the puncta, with typical vacuole-like formations suggestive of possible association with specific cellular compartments. Indeed, we observed significantly higher codistribution with LysoTracker within N370S lysosomes in PCC quantification (Fig. 4M,N). In addi- tion, Filipin IntDen. in LysoTracker-positive organelles was significantly increased in mutant cells (Fig. 4O) and not in idiopathic-PD fibroblasts (Suppl. Fig. 2C,E,F,G). This suggests that N370S mutation triggers lysosomal cholesterol accumulation.

Multilamellar Body and Autophagic Vacuole Buildup in N370S Lysosomes
To explore the mechanisms leading to lysosomal cholesterol increase, we used chloroquine (CQ), which blocks cholesterol trafficking to the ER, producing cholesterol accumulation in lysosomes.17 In control fibroblasts, CQ induced the formation of bright, peri- nuclear Filipin-positive cholesterol punctae (Fig. 4P,Q) and MLBs, composed of lipid rafts (Fig. 4R,S), similar to the pattern observed in N370S fibroblasts under basal conditions. However, CQ did not alter Filipin staining nor MLB buildup in the mutants (Fig. 4R,S). These results indicate that the MLB accumulation in N370S fibroblasts may correspond to degenerating autophagolysosomes or lysosomes, which previously built up free cholesterol among other lipids. Support- ing this idea, we observed abundant AVs containing membranous lipid storage material in CQ-treated con- trol and in basal and CQ-N370S cells (Fig. 4T, aster- isks). As shown previously, ambroxol rescues GCase1 levels and activity in N370S fibroblasts (Suppl. Fig. 1A,B,C,D). Furthermore, we also observed that ambroxol was able to decrease MLBs and AVs in N370S fibroblasts (Suppl. Fig. 1 E,F,G).

Cell Vulnerability and Oxidative Stress in N370S-PD Fibroblasts
We next investigated whether the dysfunctional autophagy-lysosome system induced by N370S muta- tion affected cell vulnerability and death. Under basal conditions, there were no differences between mutant and control fibroblasts (Fig. 5A). We treated cells with CQ to induce cell vulnerability or tert-butyl hydroper- oxide (tBHP) to induce oxidative stress. Chloroquine increased early and late apoptosis; this increase was significantly potentiated in the N370S mutants (Fig. 5A). EBSS or tBHP had little effect on cell death (data not shown), and the increase produced by EBSS plus tBHP was significantly greater in N370S cells than in controls (Fig. 5B,C), suggesting that N370S fibroblasts are more vulnerable to oxidative stress.
We evaluated reactive oxygen species (ROS) produc- tion and detected superoxide ions via DHE flow cytometry (Fig. 5D,D’). Under basal conditions, N370S fibroblasts had more superoxide ions than con- trols, and tBHP did not potentiate these levels (Fig. 5D,D’). Because ROS production may be a conse- quence of mitochondrial failure, we used DiOC6(3) to measure Dwm. Under basal conditions, we observed a significant reduction in Dwm in N370S fibroblasts (Fig. 5D’), suggesting that mitochondrial electron transport chain disruption resulted in the accumula- tion of superoxide anions. tBHP decreased Dwm in control and mutant cells, even in the absence of addi- tional ROS production in the mutants (Fig. 5D,D’). Our EM studies have highlighted mitochondrial swell- ing and alterations in the mitochondrial ultrastructure in N370S fibroblasts (Fig. 5E,F stars) that correlate with increased ROS production (Fig. 5D,D’).
To determine if the increased ROS levels in N370S fibroblasts were caused by impaired antioxidant
FIG. 4. N370S mutation causes lysosomal cholesterol, MLB, and AV accumulation, leading to lysosomal dysfunction. (A) Representative LysoTracker images of fibroblasts after treatment with EBSS, rapamycin (Rap., 500 nM), or bafilomycin-A1 (Baf-A1, 100 nM) for 4 hours. (B) Quantification of integrated LysoTracker signal intensity. Note that a higher signal in the mutants indicates more lysosomes. (C) Representative LAMP1 immunofluo- rescence images showing more lysosomes clustered close to the nucleus in mutant fibroblasts. Insets in the panels are enlarged in adjacent images. (D) Quantification of fibroblasts displaying perinuclear lysosome clustering. (E, F) Representative WB of LAMP1 in fibroblasts and its quanti- fication after Baf-A1 (100 nM) for 4 hours. (G) Basal glycohydrolase enzyme activities (b-galactosidase and b-hexosaminidase; nmol h/mg). (H,I,J) Representative WBs of cathepsin B and cathepsin D in fibroblasts and quantitative densitometry. (K) Representative plots and their quantification (L) illustrating cathepsin B activity as determined by flow cytometry. (M) Overlay of LysoTracker and Filipin fluorescence signals exhibiting greater coloc- alization of lysosomes and free cholesterol in mutants. The rightmost images illustrate the colocalization mask. (N) Quantification of Filipin and Lyso- Tracker colocalization using PCC. (O) Quantification of Filipin integrated intensity in LysoTracker-positive organelles. (P, Q) Representative images illustrating that chloroquine (CQ, 50 lM for 4 hours) increases Filipin signal in controls but not in mutant fibroblasts and their quantification. (R) Elec- tron micrographs illustrating accumulation of AVs and MLBs in mutant fibroblasts. Insets in the panels are enlarged in the adjacent images; note the typical undulation of the membrane of concentric lamellae suggesting increased storage of free cholesterol in mutant fibroblasts. Quantification of MLBs (S) and AVs (T). Clustered lysosome cells, white arrowheads; MLBs, blue arrowheads; AVs, green asterisks; Nu, nucleus. Samples in the blots were processed in parallel. Data represent the mean 6 SEM of at least 3 independent experiments from all cell lines, *P < 0.05; **P < 0.01; ***P <0.001 versus controls; #P < 0.05, ##P < 0.01; ###P < 0.001 versus untreated fibroblast, 2-way ANOVA followed by Bonferroni post hoc analysis (B, F, Q, S, T), Student t test (G, J), and Mann-Whitney U test (L, N, O) and v2 test (D). Scale bar: 10 lm, A, M, P; 20 lm, E; 0.5 lm, R.
FIG. 5. Increased ROS production, cell death, and antioxidant defense mechanisms in N370S-PD fibroblasts. (A) Flow cytometry analysis showing apoptosis in fibroblasts treated with chloroquine (CQ, 50 lM for 4 hours). (B, C) Immunofluorescence of cleaved caspase-3 staining (green) and Hoechst (blue) illustrating increased cleaved caspase-3-positive cells (arrows) in mutant fibroblasts after tert-butyl hydroperoxide (tBHP, 100 lM for 4 hours) plus EBSS treatment and its quantification is shown in C. Insets in the merged panels are enlarged in adjacent images. (D) Representative plots illustrating higher DHE intensity in mutant fibroblasts under basal conditions and quantitative analysis (D0) of DHE and DiOC6(3) showing ROS and Dwm changes, respectively, in mutant fibroblasts after tBHP treatment (100 lM for 4 hours). (E, F) Electron micrographs illustrating mitochondria (pseudocolored in red) and its quantification. Observe the fragmented and swollen (*) mitochondria in mutants. (G) Increased oxidative stress marker genes SOD1 and CAT in N370S fibroblasts. (H, I) WB analysis of SOD1 after tBHP treatment (100 lM for 4 hours). Note that tBHP increased SOD1 levels only in control fibroblasts and not in the mutants. Samples in the blots were processed in parallel; data represent the mean 6 SEM of at least 3 independent experiments from all cell lines. *P < 0.05; **P < 0.01; ***P < 0.001 versus controls; and #P < 0.05; ##P < 0.01; ###P < 0.001 versus untreated fibroblasts; 2-way ANOVA followed by Bonferroni post hoc analysis (A, D0, I), Student t test (C, G), and Mann-Whitney U test (F). Mit, mito- chondria. Scale bar: 25 lm, B; 0.5 lm, E.
FIG. 6. Schematic diagram summarizing the primary altered features found in N370S-GBA1 fibroblasts in PD. This mutation reduces GCase1 activity in the lysosomes caused by its retention in the ER, which, in turn, produces stress, enlargement, and disorganization of the ER and GA fragmenta- tion. There is more autophagosome accumulation in N370S than in the controls. p62/SQSTM1 accumulates in N370S fibroblasts because of dys- functional lysosomes likely caused by cholesterol accumulation that promotes MLB formation. These alterations favor the abnormal removal of damaged mitochondria, ROS production, and increased cell vulnerability and death. enzyme function, we measured SOD1 and CAT mRNA levels. Under basal conditions, significant increases were detected in N370S fibroblasts (Fig. 5G), as also found in SOD1 protein levels (Fig. 5H,I). In contrast, tBHP induced SOD1 only in control cells, indicating that the antioxidant system was hyperacti- vated in N370S mutant cells, although not sufficiently to scavenge ROS (Fig. 5D,D’).
Our findings suggest that N370S GBA1 mutation causes lipid accumulation and lysosomal dysfunction, triggering abnormal removal of altered mitochondria, followed by ROS production, increased cell vulnerabil- ity, and, finally, cell death.

Discussion

Although GBA1 heterozygous mutations are the most well-known genetic risk factors for PD, their impact on cellular homeostasis and their action mechanisms are not completely understood. We have shown that reduced lysosomal GCase1 levels and activity are a result of the retention of N370S GCase1 within the ER, interrupting its cellular traffic, and not to changes in transcription, extending previous studies.6,7,12,13,38,39
We have shown for the first time reduced GCase1 levels localized in the lysosomes and increased levels localized in the ER in N370S fibroblasts. Although the mechanism of GCase1 retention in the ER is not known, it is possible that the conformational instabil- ity of the mutated form impairs its binding with the transporter LIMP-2, a process that precisely occurs at the ER.40 We demonstrated disorganized ER ultra- structure and ER-stress marker upregulation in N370S fibroblasts, both of which trigger the UPR. This likely reflects dysregulation of ER homeostasis induced by misfolded GCase1. Our findings are consistent with brain postmortem analysis from GBA1-PD patients, GBA1-PD-derived dopaminergic neurons and fibroblasts, and GD models showing UPR activa- tion.6,7,41,42 Such misfolded GCase1 decreases GCase2 activity, as reported in GBA1-PD-derived cells.13,43 Because GCase2 is located in the ER, the ER perturba- tions noted above may affect its activity, suggesting the existence of crosstalk between GCase1- and GCase2-dependent metabolic pathways.
Functional GCase1 is required to maintain GA orga- nization because its ER retention induces morphologi- cal alterations in both organelles. We suggest that GA disruption could impair ER-to-Golgi protein traffick- ing, contributing to this GCase1 retention. Golgi apparatus fragmentation has been reported in other PD models as consequence of a-syn aggregation11,44 and in postmortem brain tissue from PD patients.45 However, this fragmentation appears to be a primary defect in our GBA1-PD fibroblasts because these cells present undetectable levels of a-syn (data not shown). All these results strongly suggest that misfolded GCase1 in the ER activates ER stress, UPR, and GA fragmentation, indicating that these processes play a major role in GBA1-PD pathogenesis.
Although autophagy dysregulation in GD and GBA1-associated PD has been previously reported,6,13,46 the underlying mechanisms remain unclear. N370S fibroblasts exhibited marked accumulation of autopha- gosomes and p62/SQSTM1 protein under basal condi- tions. Accumulation of p62/SQSTM1 might also be a result of reduced lysosomal autophagic clearance as well as increased autophagy activation. Similar results were reported in GBA1-N370S-derived dopaminergic neurons and fibroblasts.6,13,47 Moreover, our N370S fibroblasts exhibited decreased p-mTOR levels, as found in GBA1-deficient mice cells,10 suggesting that autophagy onset is exacerbated. Furthermore, Beclin1 was increased in our GBA1-PD fibroblasts, as in N370S PD neurons and GD fibroblasts,6,48 Therefore, the N370S mutation may not only accelerate autophago- some formation (LC3 and AV accumulation) via mTOR and Beclin1 but also impair autophagic clear- ance (p62/SQSTM1 increase) because of a secondary effect of lysosomal failure, leading to autophagosome accumulation. Consistently, we found lysosomal dys- function in N370S fibroblasts. Our fluorescence and EM analyses revealed for the first time that cholesterol accumulates in lysosomes of N370S cells, highlighting a clear relation between GBA1 mutations and free choles- terol accumulation in PD. Interestingly, GCase1 also has glucosyltransferase activity that catalyzes the for- mation of cholesteryl-glucoside (b-ChlGlc),49 support- ing the link between GCase1 function and cholesterol metabolism. Furthermore, intracellular cholesterol buildup affects ER-associated degradation during GD progression20 and alters the activity/stability of GCase1 in NPC fibroblasts.19 Our findings resemble those of patients with glycosphingolipidoses18,19 characterized by a defect in lipid trafficking within the endosomal- lysosomal system. It is possible that lysosomal choles- terol accumulation mediates changes in membrane lipid and protein trafficking either through alterations in membrane fluidity or coalescence of cholesterol- sphingolipid microdomains.18,50 Furthermore, if b- ChlGlc levels decrease because of GCase1 inhibition, the physical properties of lysosomal membranes might change and affect their function. We demonstrated that increased cholesterol storage in N370S lysosomes is characteristic of these cells and also CQ-treated control cells. Both cell types accumulated MLBs, which might be cholesterol-containing rafts in late endocytic/lyso- some organelles, an aberrant form of lipid stor- age.32,51,52 Moreover, activation of upstream steps in the autophagy pathway in N370S fibroblasts supports the role of autophagy in MLB formation.32 Our results suggest that N370S-PD and lysosomal storage lipidosis share exacerbated free cholesterol accumulation in con- centric lamellae within lysosomes. Unesterified choles- terol moves to the ER for reesterification or utilization by the cell and to the trans-Golgi network and/or plasma membrane for efflux from the cell.53 Therefore, disrupted cholesterol trafficking from N370S lysosomes may alter the membrane organization of both the ER and GA.
Lysosomal impairment appears intricately linked to PD pathogenesis,1 and high cholesterol level has been proposed as a marker of immature lysosomes.54 The sig- nificant increase in the lysosomal mass observed in our N370S-PD fibroblasts is consistent with recent studies in iPSC-derived dopaminergic neurons6,11,13 and in PD and GD fibroblasts.48,55 Notably, the reduced b-galactosidase and cathepsin B lysosomal activity in N370S-PD fibroblasts supports the idea that lysosomal function is impaired as a consequence of decreased GCase1 activity and free cholesterol accumulation. Nonetheless, unchanged b-hexosaminidase-A activity indicates that dysfunction was not complete. Our experi- ments with rapamycin and starvation revealed that lyso- somes were increased in N370S mutants, suggesting that their formation was enhanced to compensate for their failure. The presence of perinuclear lysosome clustering in N370S-PD fibroblasts supports lysosomal dysfunc- tion. Similarly, lysosome clustering was recently identi- fied in LRRK2 and GBA1-PD fibroblasts correlating with altered lysosomal Ca21 signaling.25,47
Autophagy-lysosome pathway alterations related to
changes in cellular lipid/cholesterol can inhibit degra- dation of damaged mitochondria. Our N370S fibro- blasts exhibited fragmented mitochondria with occasionally enlarged and swollen profiles similar to those found in GD mouse brains.56 These alterations suggest mitochondrial dysfunction, supported by increased ROS production and Dwm impairment, as shown in GBA1-PD12 and GD models.57
Remarkably, under basal conditions the apoptosis levels did not change in N370S-PD fibroblasts. This finding can be explained by the induction of compen- satory mechanisms such as a ROS scavenger system (CAT and SOD1), which, although it does not block ROS, protects cell viability at baseline. N370S-PD fibroblasts, however, exhibited significant sensitivity to CQ or tBHP toxicity. CQ-induced cell death may involve a severe intracellular cholesterol increase, causing lysosomal dysfunction and loss of lysosomal membrane integrity, leading to cathepsin release and cell death signaling.17 Cell vulnerability associated with N370S mutation may be caused by similar mech- anisms because our fibroblasts exhibited high choles- terol levels. In addition to these mechanisms, cell death was only observed after harsh stimuli (tBHP and CQ), suggesting that noncell autonomous mecha- nisms promote vulnerability in N370S-GBA1 cells. Whether signals coming from nonneuronal cells trigger cell death in dopaminergic neurons of PD patients is still under debate. However, that a high percentage but not all of N370S-GBA1 carriers develop PD sup- ports the implication of a noncell autonomous role for GBA1 mutations.
Our novel findings indicate that N370S mutation changes the subcellular GCase1 localization, inducing ER stress and disorganization along with lysosomal cholesterol accumulation. This could lead to altered autophagy-lysosome pathways, mitochondrial dysfunc- tion, and increased cell vulnerability (Fig. 6). Our results provide a framework for understanding the mechanisms underlying GBA1-linked PD and also sug- gest that this type of PD shares common neurodegen- erative features with lipid storage diseases.

Acknowledgments:
We thank Beatriz Pro, Emilia Rubio, Ma Jos´e Rom´an, and Mr. Martin Ian Maher for their excellent technical assis- tance. We also thank Enrique Sahagun PhD (Scixel, Madrid, Spain) for his assistance with the summary figure.

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