Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinson’s Disease

Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinson’s Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinson’s Disease Preethi Sheshadri, Maria Alicia Costa Besada, Alessia Fisher, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7558589/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Heterozygous mutations in the Glucocerebrosidase gene ( GBA1 ), which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are a genetic risk factor for Parkinson’s disease (PD), characterised by lysosomal dysfunction. The pathological effects of GBA1 mutations on PD, especially their influence on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear. Methods Fibroblasts and dopaminergic neurons, generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD, were used in the study. Live-cell imaging was performed to assess lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial cristae density and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was evaluated using FLIM-FRET, and pharmacological interventions included rapamycin and acidic nanoparticles. Statistical analyses involved unpaired t-tests, one-way ANOVA, and two-way ANOVA. Results GCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. MTORC1 was constitutively phosphorylated and FLIM-FRET measurements confirmed impaired V-ATPase assembly, which was reversed following rapamycin treatment. Rapamycin and lysosome-specific acidic nanoparticles rescued lysosomal pH, restored mitophagy and mitochondrial membrane potential in GBA1 mutant dopaminergic neurons. Conclusions Our findings identify lysosomal acidification as the primary cause of impaired bioenergetic function and reduced mitophagy in GBA1-PD. MTORC1-mediated disruption of V-ATPase assembly drives these pathogenic processes. Pharmacological interventions that restore lysosomal pH—such as rapamycin or acidic nanoparticles—rescue both lysosomal and mitochondrial defects, offering a promising therapeutic approach for GBA1-PD. GBA1 Parkinsons Disease mitochondria lysosomes lysosomal pH MTOR acidic nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Genome-wide association studies have revealed that mutations of the glucocerebrosidase ( GBA1 ) gene constitute a significant risk factor in the development of Parkinson’s Disease (PD)[ 1 ]. The GBA1 gene encodes the enzyme β-glucocerebrosidase (GCase), which generates glucose and ceramide from glucosylceramide within lysosomes. While homozygous GBA1 mutations cause Gaucher Disease, which may include a significant neurodegenerative component[ 2 ], heterozygous mutations are associated with an increased risk of developing PD. The two most prevalent GBA1 mutations associated with PD are the N370S and L444P mutations[ 3 ]. Intriguingly, the less common E326K mutation in GBA1 exhibits a relatively mild effect on GCase activity, does not cause Gaucher’s Disease, but is correlated with PD risk[ 4 ]. Mitochondrial dysfunction appears to constitute a defining characteristic of PD[ 5 ]. The adverse impact of dysfunctional mitochondrial oxidative phosphorylation (OXPHOS) on the viability of dopaminergic (DA) neurons is substantiated by experimental models of PD that result from toxins that target complex I[ 5 ]. The Gba1 mouse knockout model, a model of severe neurodegeneration, exhibited severe mitochondrial dysfunction in primary neurons and astrocytes in culture, alongside neurological pathologies associated with PD, including disruption of autophagy-lysosomal pathways, and the accumulation of ubiquitinated proteins and α-synuclein[ 6 ]. The autophagosome-lysosome axis and ubiquitin proteasome systems accomplish degradation of dysfunctional organelles and protein degradation and together play a critical role in cellular quality control[ 7 ]. Lysosomal acidification generating pH values between 4.5 to 4.7[ 8 ] is essential for normal lysosomal function and is required for the activity of lysosomal hydrolytic enzymes and effective protein degradation[ 44 , 12 ]. The acidification is generated by the vacuolar-type H + ATPase (V-ATPase), a proton pump composed of a peripheral V 1 domain, which hydrolyses ATP, and a membrane-integrated V o domain, responsible for the translocation of protons into the lysosomal lumen[ 10 ]. Disrupted V-ATPase assembly and V-ATPase dysfunction have been reported associated with multiple disorders[ 11 ], including Juvenile-onset Parkinson’s Disease[ 12 ], Alzheimer’s Disease[ 13 , 14 ], Epilepsy[ 15 , 16 ] and Down’s syndrome[ 17 ]. GBA1 -linked pathologies such as Gaucher’s disease and PD are associated with a dysfunctional autophagy-lysosome pathway, including impaired lysosomal regeneration from autolysosomes during macroautophagy[ 18 , 19 ]. These investigations suggest that targeting the dysfunctional lysosomal system and the autophagy-lysosome pathway may serve as a potential therapeutic strategy for PD linked to GBA1 mutations. Although numerous studies indicate that impaired lysosomal acidification plays a role in neuronal pathologies associated with various neurodegenerative disorders[ 38 ] and that GBA1-PD is characterised by lysosomal dysfunction [ 44 ], the effects of compromised lysosomal acidification in GBA1-PD have not been extensively examined. In this study, we have characterised the consequences of GBA1 mutations associated with PD for mitochondrial and lysosomal function in patient-derived fibroblasts and DA neurons generated from patient-derived induced pluripotent stem cells (iPSC). As GBA1 is a lysosomal enzyme, we hypothesised that lysosomal dysfunction as a consequence of the mutations, might lead to impaired mitochondrial function, initiating a pathophysiological cascade culminating in cell injury. Furthermore, we found that constitutive and inappropriate activation of mechanistic target of rapamycin complex 1 (MTORC1) contributes to impaired lysosomal acidification through aberrant assembly of the V-ATPase complex, leading to lysosomal dysfunction. Additionally, we demonstrate that the restoration of lysosomal pH ameliorates autophagy and rescues both lysosomal and mitochondrial function in cells carrying GBA1-PD mutations. Results To characterise the impact of PD-related GBA1 mutations on lysosomal and mitochondrial function, we examined three lines of human dermal fibroblasts and six iPSC lines derived from GBA1-PD patients carrying E326K or N370S mutations, along with two healthy controls. We also included one CRISPR-corrected isogenic control iPSC line each for GBA1-N370S and GBA1-E326K iPSCs. (Table 1 ). Table 1 – List of cell lines used Cell Lines used Cell Line ID Age/Sex Cell type Mutation Source Ethics Committee Approval CONTROL 1 CONTROL 1 77/Male Fibroblasts No Mutation NHNN PD Royal Free Ethics N370S ND34263 65/Male Fibroblasts; source for NH50182 iPSCs GBA1-N370S; Heterozygous NIH-RUCDR CONTROL 2 CONTROL 2 55/Male Fibroblasts; source for Control1 iPSCs No Mutation NHNN PD Royal Free Ethics E326K 1 Het1/ JS48753 58/Male Fibroblasts; source for Het1/E326K 1 iPSCs GBA1-E326K; Heterozygous NHNN PD Royal Free Ethics E326K 2 ND41015 63/Male Fibroblasts; source for ND50045 iPSCs GBA1-E326K; Heterozygous NIH-RUCDR Isogenic Control (IsoC) NH50142 63/Male iPSCs; Isogenic Control for ND50045 CRISPR corrected GBA1-E326K heterozygous mutation NIH-RUCDR E326K ND50045 63/Male iPSCs GBA1-E326K; Heterozygous NIH-RUCDR Isogenic Control (IsoC) NH50186 65/Male iPSCs; Isogenic Control for NH50182 CRISPR corrected GBA1-N370S heterozygous mutation NIH-RUCDR N370S NH50182 65/Male iPSCs GBA1-N370S; Heterozygous NIH-RUCDR Control Control 1 55/Male iPSCs No Mutation Derived from CONTROL 2 fibroblasts PD Royal Free Ethics for CONTROL 2 fibroblasts E326K 1 Het1/ JS48753 58/Male iPSCs GBA1-E326K; Heterozygous Derived from Het1 fibroblasts PD Royal Free Ethics for JS48753 fibroblasts E326K 2 Het 2/ MB240649 63/Male iPSCs GBA1-E326K; Heterozygous Derived from MB240649 fibroblasts PD Royal Free Ethics (for MB240649 fibroblasts Control SFC156 (Control 2) 65/Male iPSCs No Mutation EBiSC N370S 1 SFC834 (N370S1) 72/Male iPSCs GBA1-N370S; Heterozygous EBiSC N370S 2 SFC848 (N370S2) 68/Male iPSCs GBA1-N370S; Heterozygous EBiSC Lysosomal function is impaired in cells with PD-related GBA1 mutations As GBA1 is a lysosomal enzyme, our investigation began by characterising the lysosomes in fibroblasts and DA neurons from both control and PD patient-derived iPSCs. The GBA1 protein expression levels measured by western blot were not significantly different between the control and GBA1 mutant neurons; however, GCase enzymatic activity was significantly reduced in iPSC-DA neurons and fibroblasts carrying either GBA1 mutation (Fig. 1A, B, S1A, B). Lysosomal acidification was measured using the ratiometric pH-sensitive dye, Lysosensor Yellow/Blue DND160. These data revealed impaired acidification in both E326K and N370S DA neurons and fibroblasts (Fig. 1C, S1C, D). Calibration of the lysosomal pH (see methods) gave values of 4.74±0.03–4.77±0.02 in control cells but between 4.89±0.02 and 5.06±0.03 in the mutant fibroblasts (Fig. 1D). The function of endocytic trafficking and lysosomal proteolytic activity was measured using the DQ-Red BSA assay. DQ-Red BSA intensity was significantly reduced in the GBA1 mutant neurons, indicating impaired lysosomal degradative function in GBA1-PD cells (Fig. 1E, S1E). Autofluorescence excited at 355nm and imaged between 400-600nm showed an unusually bright extramitochondrial component in GBA1-PD fibroblasts (Fig. 1F) that was significantly greater than control cells. Spectral scanning and linear unmixing separated the expected mitochondrial NADH signal with an emission peak at 450nm and an extensive non-mitochondrial signal with a peak emission at 480nm, attributed to accumulated lipofuscin. Lipofuscins are cytoplasmic granules generated as a consequence of autophagy and phagocytosis processes, which show auto-fluorescence over a broad spectrum ranging from 480nm to 700nm when excited by ultraviolet or blue light[ 20 ]. While the accumulation of lipofuscin serves as an indicator of ageing, abnormal accumulation is consistent with impaired lysosomal acidification and defective autophagy[ 21 ]. Accumulation of lipofuscin is also a hallmark of Batten Disease (neuronal ceroid-lipofuscinoses), a severe early-onset neurodevelopmental disorder with progressive neurodegeneration also associated with lysosomal dysfunction [ 22 ]. These data collectively indicate significant lysosomal dysfunction in the GBA1-PD cells. Mitochondria are dysfunctional in cells with GBA1-PD mutations To determine whether the impaired lysosomal activity in the GBA1 mutations has a secondary impact on mitochondrial form and function, we measured mitochondrial membrane potential (ΔΨm) using the potentiometric fluorescent reporter tetramethyl-rhodamine methyl ester (TMRM). ΔΨm was significantly reduced in E326K and N370S GBA1 mutant DA neurons and fibroblasts (Fig. 2A, S2A, C). The lower ΔΨm was accompanied by mitochondrial fragmentation in the N370S and E326K DA neurons and fibroblasts (Fig. 2Aii c,d, S2Aii b). Ultrastructure analysis using electron microscopy revealed significant differences in mitochondrial morphology in the GBA1 mutant neurons, including reduced cristae density and increased mitochondrial area, indicating mitochondrial swelling in the GBA1 mutant iPSC DA neurons (Fig. 2B). Measurements of oxygen consumption rates using the ‘Seahorse’ respirometry system revealed ~ 40% decrease in basal oxygen consumption rate in the GBA1 mutant DA neurons with the isogenic controls (Fig. 2C). To exclude changes in mitochondrial mass and to explore the substrate dependence of mitochondrial respiration, we used mitochondria isolated from the cultures. This allowed measurements of respiratory rates with different substrates, favouring complex I (pyruvate/malate) or complex II (rotenone/succinate). ADP-stimulated respiration (State 3) rate using both CI and CII-dependent substrates was significantly reduced in the GBA1 mutant neurons compared to the control neurons (Fig. 2D, S2C). Western Blots of the OXPHOS complex proteins using a cocktail of antibodies to respiratory chain proteins, revealed a significant increase in the expression of Complex IV in E326K DA neurons (Fig. 2Eii a and S2Dii a) and increased expression of Complex I proteins in N370S neurons compared to the isogenic controls (Fig. 2Eii b and S2Dii b). Expression levels of the other respiratory chain proteins were unaltered in the E326K and N370S DA neurons. None of these data suggested any defect in complex I assembly or function. These data collectively point to impaired mitochondrial bioenergetic function in fibroblasts and neurons carrying GBA1 mutations. Mitophagy is impaired in cells with GBA1-PD mutations We then set out to explore the underlying mechanisms that link impaired lysosomal function to impaired mitochondrial bioenergetics in the GBA1-PD fibroblasts and DA neurons. A logical mechanism linking impaired lysosomal function with mitochondrial dysfunction might operate through dysfunctional mitophagy and our earlier work also established dysfunctional mitophagy in Gba1 KO mice[ 6 ]. We quantified mitophagy using the dual excitation probe mt-Keima (Fig. 3A). mt-Keima is a pH-sensitive probe that measures the fraction of mitochondria in neutral (pH 7–7.8 in the mitochondrial matrix) versus acidic pH (pH 4.5–4.7 in lysosomes) environment[ 23 ]. The mt-Keima signal ratio was significantly reduced in fibroblasts and DA neurons in the fibroblasts and DA neurons carrying both the GBA1-N370S and GBA1-E326K mutations indicating impaired mitophagy (Fig. 3Aii. S3Aii, Bii). Western blotting to quantify the status of autophagy pathways in the GBA1 mutant neurons revealed increased LAMP1 levels in E326K and N370S neurons, along with increased MTORC1 phosphorylation in the two mutant types. While LC3 flux, and expression of p62 and TOM20 were increased in the E326K mutant iPSC DA neurons, these were not significantly altered in the N370S DA neurons (Fig. 3B). Since the lysosomal pH is increased in GBA1-PD cells (Fig. 1D), the reduced mt-Keima signal in GBA1-PD cells could be attributed either to the altered lysosomal pH or to the impaired fusion of autophagosomes to lysosomes. To address this, we performed triple staining of fibroblasts against citrate synthase (CiS, for mitochondria), LAMP1 (for lysosomes), and LC3 (for autophagosomes). Subsequent imaging revealed that although there was no significant difference in mitophagosome (mitochondria and autophagosome colocalisation) density between GBA1 mutant and control fibroblasts, mitolysosome (colocalisation of mitochondria and lysosomes) density was significantly increased in the GBA1 mutant fibroblasts (Fig. 3C). Characterisation of electron micrographs for specific autophagic vesicle types based on Neikirk et al.,2023[ 24 ], showed that autophagic vesicles, specifically the autolysosome and lysosome number, were significantly increased in both GBA1 mutant fibroblasts and neurons. (Fig. 3Dii, S3Cii a and b). From the micrographs, the autophagosomes also appeared to be fully enclosed, ruling out incomplete phagophore formation as the underlying mechanism of autophagy defect. These data indicated that mitophagy is significantly impaired in cells carrying the GBA1-PD mutations and the decrease in mitophagy is likely due to impaired pH and not improper phagophore formation. V-ATPase complex formation is impaired in GBA1-PD Since our data showed an accumulation of lysosomes and autolysosomes in GBA1-PD fibroblasts and iPSC-DA neurons, we suspected this was a result of impaired lysosomal acidification. Assembly of the pH regulatory component in lysosomes – the Vacuolar-type H + ATPase (V-ATPase) is regulated by MTORC1[ 25 ], which was constitutively phosphorylated in the GBA1-PD mutant cells. Ratto et al.[ 25 ] demonstrated that under nutrient-rich conditions, MTORC1 is distributed in the cytosol, enabling the peripheral ATP6V 1 to bind to the membrane-bound ATP6V 0 domain, forming a functional V-ATPase complex allowing proton exchange and acidification of the lysosomes. Upon nutrient deprivation, MTORC1 is phosphorylated and remains on the lysosomal membrane, preventing the formation of a functional V-ATPase complex[ 25 ]. While the V-ATPase maintains lysosomal pH, disruption of the complex does not hamper autolysosome formation[ 26 ]. Total protein estimation through western blotting revealed no significant difference in the expression of ATP6V 0 D2 and ATP6V 1 A or ATP6V 1 H between the control and mutant DA neurons (Fig. 4A). To examine their expression levels in lysosomes specifically, we performed a lysosomal enrichment assay and probed for pMTORC1, LAMP1, and the ATP6V 0 and ATP6V 1 components (Fig. 4Bi). pMTORC1 expression was increased up to two-fold, and LAMP1 expression was increased ~ 1.5 fold in the GBA1-mutant DA neurons. However, ATP6V 1 A expression was reduced by ~ 40% and ATP6V 1 H by ~ 15% within the lysosomes fractionated from GBA1 mutant DA neurons, while ATP6V 0 D2 levels remained unchanged between the control and mutant iPSC-DA neurons (Fig. 4Bii). Furthermore, overnight treatment of GBA1 iPSC-DA neurons with the MTORC1 inhibitor rapamycin (200nM) decreased the pMTORC1 and LAMP1 levels, while increasing ATP6V 1 A and ATP6V 1 H expression in the lysosomes and the ATP6V 0 D2 levels remained constant (Fig. 4Bi b and Bii). In order to assess the V-ATPase assembly, we transfected the cells with ATP6V 1 B2-mNeongreen and ATP6V 0 a3-mScarlet and quantified their FRET interaction using fluorescence lifetime imaging microscopy (FLIM)[ 27 ]. The data were best fit to a biexponential decay function. The ATP6V 0 a3 construct utilises the more rapidly maturing but photophysically heterogeneous acceptor variant mScarlet-I[ 28 ]. This exhibits at least two fluorescence lifetimes, meaning the biexponential decay of the donor is an oversimplification given the likelihood of contrasting FRET rates to each acceptor species [ 29 , 30 ]. The results are therefore presented as the mean (amplitude weighted) fluorescence lifetime -τ m - which will nevertheless respond to variations in FRET without having to interpret the meaning of individual decay components and amplitudes. t m of ATP6V 1 B2-mNeongreen averaged 617.72± 25.3 ps in control fibroblasts but increased to 760.88± 20.36 ps in N370S and 811.03± 17.04 ps in E326K mutant fibroblasts, indicating an increased distance between ATP6V1B2-mNeongreen and ATP6V0a3-mScarlet signalled by a reduction in FRET. Overnight treatment with 200nM rapamycin significantly decreased t m to 652.7± 19.69 ps and 726.05± 21.2 ps in E326K and N370S fibroblasts respectively while the t m control fibroblasts was at 590.5± 32.7 ps (Fig. 4C). To validate the subcellular localisation process within a condensed timeframe, we administered 1 µM rapamycin to the cells and performed FLIM every 10 minutes for 40 minutes. The mean fluorescence lifetime of ATP6V1B2-mNeongreen exhibited a progressive decline in the GBA1 mutant fibroblasts, decreasing from ~ 850 ps at 0 min to ~ 580 ps at 40 minutes, while t m in control fibroblasts showed only a small reduction, decreasing from ~ 675 ps at 0 min to ~ 588 ps at 40 minutes (Fig. 4Dii). The FLIM-FRET data thus confirmed impaired assembly of the V -ATPase complex in GBA1 mutant cells. All these data are consistent with a model in which phosphorylation of MTORC1 at the lysosome membrane limits the formation of a functional V-ATPase complex in the GBA1-PD cells. Employing rapamycin as a potent MTORC1 inhibitor reduced MTORC1 activity and facilitated the formation of a functional V-ATPase complex. Acidification of lysosomes is sufficient to restore lysosomal, mitochondrial function and mitophagy in GBA1-PD Our data show that GBA1 mutations lead to significantly impaired lysosomal acidification and mitophagy and suggest that this failure of cellular homeostasis may underlie the mitochondrial dysfunction seen in GBA1-PD cells. We therefore wondered whether restoring lysosomal pH could rescue these defects in GBA1-PD cells. Since impaired V-ATPase complex formation may be a consequence of MTORC1 hyperphosphorylation, we explored the impact of treatment with rapamycin (overnight treatment with 200 nM) on lysosomal and mitochondrial function. As an independent pH modulator, we employed novel poly(ethylene tetrafluorosuccinate-co-succinate) nanoparticles (NPs), which acidify lysosomes[ 31 ]. We first validated the effects of rapamycin and the nanoparticles in patient-derived fibroblasts (Fig S4). To confirm that the NPs were localised to the lysosomes, we loaded the fibroblasts with rhodamine-tagged NPs and stained the cells with Lysotracker blue DND22 (Fig S4A) which confirmed the localisation of the NPs to lysosomes. Overnight treatment with 180 µg/mL acidic NPs composed of poly(ethylene tetrafluorosuccinate-co-succinate) restored lysosomal pH in GBA1 mutant cells to levels comparable to those of control fibroblasts as measured using the pH-sensing ratiometric probe Lysosensor Yellow/Blue DND160. Treatment with non-acidic control NPs did not significantly affect the lysosomal pH in the GBA1 mutant fibroblasts (Fig. S4B, C). TMRM staining demonstrated that both rapamycin and the acidic NPs significantly rescued the ΔΨm in the mutant fibroblasts (Fig S4F, G). We also found that the ΔΨm was increased in control 2 and E326K fibroblasts upon control NP treatment (Fig S4G). We wondered whether the effect of control NPs on ΔΨm could be due to the succinate component in the poly(ethylene succinate) control NPs. However, treatment with 3mM Diethyl Succinate for 30 minutes did not have any effect on the ΔΨm in control and GBA1 mutant fibroblasts (Fig S4H). We then assessed the impact of rapamycin and acidic NPs on GBA1 mutant iPSC-derived DA neurons (Fig. 5). Treatment with 200nM rapamycin and 180 µg/mL acidic NPs restored lysosomal pH, ΔΨm, and mitophagy in the E326K and N370S iPSC DA neurons (Fig. 5A, B, C). Although there was no change in GBA1 expression levels, GCase activity was increased in GBA1 mutant DA neurons following treatment with either rapamycin or acidic NPs (Fig S4 D, E). Additionally, western blotting of OXPHOS complexes indicated that the reduced mitochondrial Complex IV levels in E326K DA neurons and the reduced Complex I levels in the N370S DA neurons were normalised upon treatment with either rapamycin or acidic NPs. (Fig S4I). As rapamycin restores the assembly of the V-ATPase complex in GBA1 mutant cells (Fig. 4D-F), we asked whether the acidic NPs also function through the V-ATPase complex formation. FLIM-FRET experiments conducted on control and acidic NP-treated GBA1 mutant fibroblasts transduced with ATP6V 1 B2-mNeongreen and ATP6V 0 a3-mScarlet plasmids demonstrated a further increase in the t m of ATP6V1B2 in both control and mutant fibroblasts treated with acidic NPs, confirming that the acidic NPs operate independently of V-ATPase (Fig S4J). Thus, the data presented here suggest that constitutive phosphorylation of MTORC1 in GBA1-PD cells leads to impaired lysosomal pH and compromised lysosomal proteolytic function. This results in the accumulation of mitolysosomes and impaired mitophagy. The compromised mitophagy culminates in mitochondrial dysfunction in GBA1 mutant cells. Furthermore, inhibiting MTORC1 activity or pharmacologically increasing lysosomal pH using acidic NPs enhances lysosomal proteolytic activity, rescuing mitochondrial dysfunction in GBA1 mutant iPSC-DA neurons and fibroblasts. Discussion We report a range of abnormalities in the lysosomes and mitochondria of GBA1-PD fibroblasts and iPSC-DA neurons carrying E326K or N370S GBA1 mutations. Given that GBA1 is a lysosomal resident enzyme, we hypothesised that mitochondrial dysfunction is likely to reflect a primary lysosomal defect. We used the ratiometric probe Lysosensor Yellow/Blue DND 160 to measure lysosomal pH. When calibrated, these measurements revealed that lysosomal pH in control cells ranged from 4.74 ± 0.03 to 4.77 ± 0.02, while in mutant cells it showed a significant shift to a range of 4.89 ± 0.02 to 5.06 ± 0.03. Reports in the literature indicate that the optimal pH for lysosomal function ranges between 4.2 and 4.8, depending on the cell type [ 8 , 32 ]. Although the difference between control and mutant cells seems modest, the data we present suggest that this difference in mutant cells is functionally significant. In line with other studies, we observed a pH-dependent decrease in the lysosomal enzyme activity, an increase in lysosomal number, and disruption of mitophagy and mitochondrial metabolism [ 18 , 33 – 39 ]. Very few reports discuss mitochondrial abnormalities associated with GBA1-PD [ 35 , 40 – 43 ], and even fewer discuss mitophagy [ 44 ]. We noted reduced ΔΨm and mitochondrial fragmentation alongside swollen mitochondrial structures, decreased cristae density, and a reduced oxygen consumption rate, all indicating dysfunctional mitochondrial oxidative phosphorylation. This was also associated with reduced mitophagy in the E326K and N370S GBA1-PD fibroblasts and iPSC-DA neurons. Numerous studies have suggested mechanistic evidence for a connection between PD and GBA1 mutations. For example, dysfunctional mitochondria in primary neurons and astrocytes were described in the Gba1 mouse knockout model, associated with an impaired autophagy-lysosomal pathway, resulting in the accumulation of ubiquitinated proteins and α-synuclein[ 6 ]. Various studies have explored the mechanisms behind the PD pathology in GBA1 mutant models. A recent study emphasised that decreased GCase activity in GBA1-PD midbrain dopaminergic neuronal cells leads to defective modulation of the untethering protein TBC1D15, which regulates Rab7 GTP hydrolysis for contact untethering. This impairment results in prolonged mitochondria-lysosome contacts, potentially affecting mitochondrial dynamics and function in GBA1-linked PD[ 42 ]. Baden et al. proposed an alternative function of GBA1 in the mitochondria in maintaining the integrity of complex I and support of energy metabolism[ 40 ], although in the present study we found no evidence for mitochondrial localisation of GBA1 nor impaired assembly of complex I. Our respirometry assays using isolated mitochondria revealed a marked reduction in both maximal respiratory capacity and ATPase-linked oxygen consumption in GBA1-E326K and N370S mutant DA neurons, upon stimulation with complex I (pyruvate/malate) and complex II (rotenone/succinate) substrates. The impairment across both complexes points to a global mitochondrial dysfunction, potentially arising from structural abnormalities such as mitochondrial enlargement and decreased cristae density—features observed in these mutants—or as a downstream consequence of lysosomal dysfunction. Notably, lysosome-related mitochondrial impairment has also been reported in Tre-2/Bub2/Cdc16 (TBC1) domain containing Kinase (TBCK) encephaloneuronopathy, supporting a broader link between lysosomal defects and mitochondrial pathology[ 45 ]. Recent evidence suggests that the primary cause of the pathologies associated with lysosomes and mitochondria in PD is the GBA1 mutation. There appears to be a correlation between the onset of PD and lysosomal dysfunction caused by impaired GCase activity. Dysfunctional lysosomes disrupt cellular signalling networks, metabolic processes, autophagy, and the regulation of calcium (Ca²⁺) signalling, all of which may contribute to PD pathology[ 7 , 10 , 19 , 36 , 38 ]. Furthermore, both lysosomal function and autophagy are intrinsically dependent on lysosomal acidification, which is influenced by the pH-dependence of lysosomal enzyme activity and substrate degradation, as well as the regulation of lysosomal calcium efflux[ 46 ]. Li et al. also suggest that autophagy in L444P mutant GBA1-PD neurons is impaired at two stages: the initial autophagy stage and the lysosomal degradation stage. While these reports indicate a relationship between GBA1 mutations and autophagic, lysosomal, and mitochondrial dysfunction, a mechanistic dissection of these multi-organellar dysfunctions is lacking. Dysregulation of the MTORC1 pathway is reported in various neurodegenerative diseases, including PD and Alzheimer’s disease[ 47 , 48 ]. A recent study by Chen et al. suggests that hyperphosphorylation of MTORC1 in striatal inhibitory neurons diminishes the disruption of dopamine receptor activity, alongside odour preference, in transgenic mice [ 49 ]. Liu et al. demonstrate the role of MTORC1 in dopamine dynamics and synaptic plasticity[ 50 ]. These results elucidate the additional impacts of increased phosphorylated MTORC1 on PD. Kaempferol, an MTORC1 inhibitor, has also been shown to promote autophagy and to protect DA neurons in the MPTP-induced C57BL/6J–PD mouse model [ 51 ]. All this literature suggests that hyperphosphorylated MTORC1 may play a central role in the development of PD associated with GBA1 mutations. Our study also indicates increased MTORC1 phosphorylation in GBA1-PD fibroblasts and iPSC-DA neurons. While the link between MTORC1 hyperphosphorylation and GBA1 mutations is unclear, it may reflect a cellular stress response to lipid substrate accumulation, indicating nutrient deprivation under GBA1 mutant conditions[ 52 – 54 ]. Recent evidence underscores the significance of impaired lysosomal acidification in various disease processes. Notably, impaired lysosomal acidification is proposed to be a critical factor in the pathogenesis of multiple neurodegenerative diseases[ 13 , 24 , 27 , 56 ]. Mutations in presenilin-1 ( PS1 ) associated with familial Alzheimer’s disease have been demonstrated to inhibit the lysosomal acidification of human PS1 mutant fibroblasts, resulting in chronic alterations in autophagy and degradation, which may be restored through lysosomal reacidification[ 55 ]. Furthermore, it has been established that individuals with Down Syndrome, who are predisposed to early-onset dementia resembling Alzheimer’s disease, exhibit increased levels of the β-cleaved carboxy-terminal fragment of the amyloid precursor protein due to the presence of an additional chromosome 21, which impairs lysosomal acidification and functionality via the inhibition of V-ATPase[ 17 ]. Collectively, these studies illuminate the connections between the impairment of lysosomal acidification in autophagic pathways and the pathogenesis of neurodegeneration, thereby presenting potential therapeutic avenues for targeting lysosomal function and pH to mitigate these debilitating diseases. Acidic NPs in the form of poly(lactic-co-glycolic acid) (PLGA) acidic NPs have been previously used to reduce lysosomal pH and disease progression in cells derived from GBA1-N370S PD patient fibroblasts and in PD mouse models[ 57 ]. Additionally, poly-succinate acidic NPs used in this study have been employed to restore lysosomal pH and rehabilitate metabolic and lysosomal functions in cases of non-alcoholic fatty liver disease[ 31 ]. As an MTORC1 inhibitor[ 58 ], rapamycin also acts as a prominent inducer of mitophagy and has been demonstrated to mitigate mitochondrial dysfunction in various disease models through inhibition of the MTOR pathway[ 59 , 60 ]. Consistent with findings from other studies, we found that rapamycin rescues mitophagy and mitochondrial function in GBA1-PD fibroblasts and DA neurons as well. This could either be due to the role of MTOR as a regulator of mitochondrial function[ 61 , 62 ] or a downstream effect of lysosomal acidification by MTOR inhibition. To establish whether impaired lysosomal acidification is the cause of these dysfunctions, we employed an independent pH modulator in the form of novel acidic poly-succinate NPs, which rectify lysosomal pH in the cells while enhancing lysosomal and mitochondrial functions in GBA1-PD fibroblasts and DA neurons. Our data confirmed the efficacy of acidic NP treatment on lysosomal pH in vitro . We show that the ratio of alkaline vs acidic lysosomes decreased significantly in all mutants and most controls after the acidic NP treatment, demonstrating a decrease in mean lysosomal pH. The effect on controls could be due to the alkalinisation of the lysosomes with age, which can be reversed by the acidic NP treatment in healthy cells[ 63 ]. Importantly, treatment with acidic NP successfully eliminated the differences in lysosomal pH between the GBA1 mutants and the controls. We conclude that the restoration of lysosomal acidification in the mutant cells improved lysosomal function. Lysosomal acidification also rescued GCase activity in the cells despite mutations in the GBA1 protein. Other studies have also demonstrated the restoration of GCase activity despite GBA1 mutations under acidic pH conditions[ 64 – 66 ]. The N370S mutation results in a structurally rigid protein with reduced intralysosomal stability and reduced GCase activity[ 66 ]and the E326K mutation has little effect on the active catalytic site of the protein but affects the dimerisation and quaternary structure formation due to folding defects, likely leading to a mild reduction in GCase activity at higher pH levels[ 64 ]. GCase activity depends on protonation of amino acids at the active sites and due to interaction of the protein with activators like saposin C[ 67 ], which is affected due to the mutation[ 64 , 68 ]. Restoring the acidic pH in the lysosomes helps restore functional GCase activity despite the mutations possibly by stabilising protein structure, improving folding and optimising the active-site ionisation[ 64 , 69 ] To explore whether altered lysosomal pH is related to mitochondrial dysfunction in GBA1-PD, we evaluated ΔΨm, redox levels, and oxidative phosphorylation (OXPHOS) protein levels following treatment with NPs. Our results reveal a significant rescue of ΔΨm among GBA-PD mutants after acidic NP treatment, indicating improved mitochondrial function. When compared to untreated healthy controls, the difference in ΔΨm was no longer statistically significant in the mutants following treatment with acidic NPs. It is noteworthy that some control samples also exhibited alterations due to acidic NP treatment, resulting in a small but significant increase in ΔΨm. We also found a significant increase in ΔΨm in control 2 and E326K fibroblasts upon control NP treatment. Since the NPs are composed of poly(ethylene-succinate), we suspected this could be a substrate-mediated (succinate) effect in control NP treated conditions. However, 30-minute treatment with 3mM Diethyl Succinate did not alter the ΔΨm in the control or mutant fibroblasts. Rescuing lysosomal pH also improved mitophagy in the GBA1-PD fibroblasts and iPSC-DA neurons. Conclusions We have explored the relationship between lysosome biology, pH, mitophagy and mitochondria in cells carrying PD-associated mutations of GBA1 . A detailed examination of the pathophysiology of these cells reveals a range of abnormalities in the lysosomes, including reduced GCase activity, an increased lysosomal number, impaired lysosomal acidification, and impaired pH-dependent lysosomal proteolytic activity. Mitochondrial dysfunction is reflected by reduced mitochondrial membrane potential, swollen, fragmented mitochondria, decreased cristae density, and the accumulation of abnormal mitochondria, along with a reduced oxygen consumption rate. We also found a decrease in mitophagy and an accumulation of autolysosomes in the GBA1-PD cells. We show that increased pMTORC1 on the lysosomal membrane, possibly due to accumulation of substrates in the lysosomes, causing nutrient deprivation, hinders the formation of a functional V-ATPase complex in GBA1-PD cells, thereby compromising lysosomal acidification. We found that inhibiting MTORC1 using rapamycin rescues functional V-ATPase formation and lysosomal pH in the GBA1-PD neurons and rescues mitophagy and mitochondrial function. Independent modulation of lysosomal pH using acidic poly-succinate NPs, restored lysosomal pH, rescued mitophagy and restored mitochondrial function in the GBA1-PD fibroblasts and iPSC-DA neurons (Fig. 6). Since rapamycin has broad effects, the comparison to NPs is valuable, adding a translational value for future applications. We thus unravel the mechanism behind impaired lysosomal pH in GBA1-PD cells and demonstrate that restoring lysosomal pH is sufficient to rescue both lysosomal and mitochondrial dysfunction, along with mitophagy in GBA1-PD fibroblasts and iPSC-DA neurons. Methodology Cell lines and ethics committee approval: The human iPSC (hiPSC) and fibroblast lines used in the study, along with their sources, are detailed in Table 1 . Fibroblasts were cultured in DMEM (1x) + GlutaMAX™ basal media (Gibco 2206106), 10% (v/v) Foetal Bovine Serum (Gibco 10270098), and 1% antibiotic-antimycotic (Gibco 15240096), and incubated at 37 ºC with 5% CO 2 . The media was changed every 72 hours, and the cells were passaged upon reaching 80% confluency using 0.25% Trypsin-EDTA (Gibco 25200056). Passages between 9 and 15 were used for experimental purposes. Human GBA1 patient-derived iPSC lines were cultured under feeder-free conditions on Geltrex-coated 6-well plates, using mTeSR Plus medium and were passaged every 4–5 days with a 0.5 mM EDTA solution or upon reaching 80% confluency. Passage numbers between 20 and 30 were employed for differentiation purposes. Human pluripotent stem cell-derived DA neurons DA neurons were generated from iPSCs using a step-by-step differentiation protocol, as previously described[ 70 ]. Briefly, 90–95% confluent hiPSCs were cultured on Geltrex-coated plates and maintained in N2B27 medium, which is a 1:1 mix of Neurobasal (Gibco, 21103049) supplemented with 1X B27 (Gibco, 17504044), Glutamax (Gibco, 35050-038), and 0.5X Anti-anti (Gibco, 15240096), and DMEM-F12 (Gibco, 10565018) supplemented with 1X N2 supplement (Gibco, 17502048), 1X MEM Non-Essential Amino Acids (Gibco, 11140-050), 0.5X Anti-anti (Gibco, 15420096), 50 µM β-mercaptoethanol (Gibco, 21985-023), and human Insulin Solution (Sigma, I9278). Daily media changes were conducted for the first 14 days. On day 0, the media was supplemented with 5 µM SB431542 (Tocris Bioscience, 1614/10), 2 µM Dorsomorphin (Tocris Bioscience, 3093/10), and 1 µM CHIR99021 until day 2. From day 2 to day 7, 1 µM Purmorphamine (Merck Millipore, SML0868) was added to the mix. From day 8 to day 14, CHIR99021 and SB431542 were removed from the medium, and the cells were maintained with Dorsomorphin and Purmorphamine in N2B27. Cells were dissociated with 1 mg/mL Dispase (Gibco, 17105041) on days 4 and 14. After patterning for 14 days, the midbrain DA neuronal precursor cells (NPCs) were maintained in N2B27 medium until day 18. On day 19, the cells were dissociated with Accutase (Gibco, A1110501) and plated onto Geltrex-coated plates at a density of 2 × 10^5 cells/cm² and terminally differentiated with N2B27 medium supplemented with Compound E (Enzo Lifesciences, ALX-270-415-C250) and 10 µM Rho-kinase inhibitor, Y-27632 dihydrochloride (Tocris, 1254) from day 20 until day 70–75, with media changes twice a week. DA neurons between days 70 and 75 were used for downstream experiments. Nanoparticle, rapamycin and diethyl succinate treatment Control and acidic NPs, composed of poly(ethylene-succinate) and poly(ethylene tetrafluorosuccinate-co-succinate) respectively, were obtained from Prof. Mark Grinstaff and Prof. Orian Shirihai. The synthesis of the nanoparticles is described in [ 31 ]. The nanoparticles were stored at -80°C in aliquots and thawed fresh prior to treatment. Aliquots of 75 mg/mL nanoparticles were thawed to room temperature, vortexed thoroughly, and added to the cells at a concentration of 180 µg/mL in fresh culture medium for 12–16 hours before the experiment. A stock solution of 200 µM rapamycin (Sigma, R0395) was prepared in DMSO and sterile filtered before being stored as aliquots at -20ºC. This stock solution was diluted to 200 nM in medium before being added to the cells. The cells were treated with 200 nM rapamycin for 12–16 hours before proceeding with the experiments unless mentioned otherwise. Diethyl Succinate (Merck, 112402) was diluted to 3 mM in culture media and sterile-filtered and added onto cells 30 minutes prior to measuring the ΔΨm using TMRM. Generation of lentivirus and transduction: pLV-Ef1a-3xHA-ATP6V1B2-mNeonGreen and pLV-Ef1a-3xFlag-ATP6Voa3-mScarlet plasmids were kindly provided by Dr. Wilhelm Palm (EPR 462 and EPR 475, European Plasmid Repository). pHAGE-mt-mKeima was a generous gift from Prof. Richard Youle (131626, Addgene). Lentiviral plasmids were packaged using psPAX2 and pMD2.G in HEK293T cells with Xtremegene-HP transfection reagent (Merck, 6366236001), according to the manufacturer’s instructions. The media supernatant containing viruses was collected 48 and 72 hours post-transfection and concentrated using Lenti-X transfection reagent (Takara, 631231) with overnight incubation, followed by centrifugation at 2000 rpm for 1 hour at 4ºC. The pellet was then resuspended in DMEM-F12 medium and stored at -80ºC for further use. 72–96 hours prior to imaging, fibroblasts or DA neurons were treated with lentiviral particles. 24 hours post-treatment, the media was replaced with regular culture medium and incubated for another 48 hours before imaging. Confocal imaging DMEM-F12 medium (Gibco, 21041025) or BrainPhys medium (Stem Cell Technologies, 05796) served as a recording buffer for live cell imaging of fibroblasts and DA neurons. All images were acquired using a Carl Zeiss LSM 880 Confocal Laser Scanning microscope with Zen Black Software and a 63×/1.40 oil immersion lens at 37°C. Fixed and stained fibroblasts were also imaged with the same equipment. Lysosensor Yellow/Blue DND 160 The cells were washed with DPBS and incubated for 4 minutes in the recording buffer containing 3 µM Lysosensor Yellow/Blue DND 160 (Invitrogen, L7545) at 37°C. After incubation, the dye was washed twice with PBS and imaged in fresh recording buffer using the UV laser at 355 nm. The dual emission maxima of the dye (440 nm/540 nm) were detected using spectral scanning (lambda scan mode) on the Zeiss LSM 880. Images were subsequently linearly unmixed employing the ZEN Black software to separate the 440 nm and 540 nm emission images. The ratio of 440/540 nm was then calculated using the Ratio Plus plugin on ImageJ/Fiji software to determine the lysosomal acidity of fibroblasts and DA neurons Lysosomal pH measurement The lysosomal pH was measured using Lysosensor Yellow/Blue DND-160 (L7545, Invitrogen). Fibroblasts cultured on fluorodishes were treated with pH calibration buffer containing Monensin (Sigma, M5273) and Nigericin (Sigma, N7143), with a pH ranging from 3.5 to 7.0 and stained with Lysosensor Yellow/Blue DND 160. The cells were incubated with 2 µM for 5 minutes at room temperature (RT) and imaged as previously mentioned. The 440/540 fluorescence ratio was measured for each calibration buffer. A lysosomal pH calibration curve was established by correlating the 440/540 nm ratio with the corresponding pH values using ImageJ/Fiji software. Measurement of lysosomal proteolytic activity by DQ Red BSA The DQ-Red BSA trafficking assay dye (Invitrogen, D12051) was utilised to examine lysosomal proteolytic activity. DA neurones were washed with DPBS, and pre-warmed medium containing DQ-Red BSA was added to the fluorodishes. The dishes were incubated at 37°C for three hours. Following the incubation period, the cells were washed with DPBS and replaced with the recording buffer prior to imaging. Cells stained with DQ-Red BSA were excited using a 561 nm Argon laser, and the emitted fluorescence was collected within the 564–740 nm range. The fluorescence intensity was quantified per cell using ImageJ/Fiji software with consistent threshold settings across all samples. Measurement of ΔΨm using TMRM Fibroblast or DA neurons were washed with DPBS and incubated in recording buffer containing 20 nM TMRM (Thermofisher Scientific, T668) at 37°C for 30 minutes. Following incubation, fresh recording buffer containing 20 nM TMRM was added before proceeding with imaging. The cells were excited for TMRM fluorescence at 561nm, and images were acquired as Z-stacks. Maximum intensity projection images were then utilised to quantify the fluorescence intensity using ImageJ/Fiji software with consistent threshold settings across all samples. The TMRM images were also used to analyse the mitochondrial morphology [ 71 ]. Measurement of Lipofuscin levels Lipofuscins are excited at 355 nm and fluoresce across a range from 480 to 700 nm[ 20 ]. Fibroblasts were washed with the recording buffer and imaged using UV illumination at 355 nm, with images obtained through spectral scanning. The images were subsequently linearly unmixed using ZEN Black software to separate the 460 nm and 480 nm emission images, thus distinguishing NAD(P)H from lipofuscins. The spectrally unmixed images at 480 nm were then utilised to calculate lipofuscin density per cell using ImageJ/Fiji software. Colocalization of the Rhodamine-B labelled aNPs with the lysosomes Rhodamine B-labelled acidic nanoparticles were treated for 12–16 hours prior to imaging the fibroblasts. On the imaging day, cells were washed with the recording buffer and incubated with 50 nM LysoTracker Blue DND-22 dye (Invitrogen, L7525) for 2 hours at 37°C. Following the incubation, the cells were washed with the recording buffer and imaged in fresh buffer. Images were acquired using sequential excitation (405 nm for LysoTracker Blue and 561 nm for Rhodamine) and emission ranges set to 420–480 nm and 570–620 nm, respectively. Colocalisation Pearson’s coefficient was quantified using ImageJ/Fiji software with consistent threshold settings across all samples. Measurement of mitophagy using mt-Keima reporter Measurement of mitophagy was performed as previously described[ 72 ]. Untreated fibroblasts and DA neurons were tranduced with lentiviral mt-Keima particles 72 hrs prior to imaging. 12–16 hours before imaging, the cells were treated with acidic nanoparticles or rapamycin. The cells were imaged using two sequential excitation wavelengths (458 nm for green fluorescence and 561 nm for red fluorescence) with an emission range of 570–695 nm. The laser power was set at the minimum output to allow the clear visualization of the mt-Keima signal. The ratio of the high F 543 :F 458 ratio values were generated using the Ratio Plus plugin in ImageJ/Fiji and was used as an index of mitophagy. Fluorescence lifetime imaging microscopy (FLIM) quantification of ATP6V 1 B2-mNeonGreen Fluorescence lifetime imaging was conducted using single-photon excitation on a multimodal time-resolved fluorescence microscope as previously described[ 73 ]. This setup encompassed an 80 MHz, near-infrared, femtosecond excitation source (Insight X3, Spectra Physics, Crewe, UK), a second harmonic generation unit (Harmonixx SHG, APE, Berlin, Germany), a laser scanning unit (DCS-120, Becker & Hickl, Berlin, Germany), an inverted microscope (Axio Observer 7, Zeiss, Cambridge, UK) featuring a high numerical aperture objective (Plan-Apochromat 63x/1.4 Oil M27, Zeiss, Cambridge, UK), an ultrafast hybrid detector (HPM-100-07, Becker & Hickl, Berlin, Germany), and time-correlated single photon counting (TCSPC) electronics (SPC-180NX, Becker & Hickl, Berlin, Germany). Images were acquired using 473 nm excitation to minimise the ratio of acceptor to donor excitation, along with 500–540 nm emission filtering to isolate fluorescence from the mNeonGreen donor. Photon counts were acquired for two minutes and histogrammed at 14.6ps intervals. Curve fitting analysis was performed in SPCImage (Becker & Hickl, Berlin, Germany). Lysosomal enrichment assay A lysosomal enrichment assay was conducted using the Lysosome Enrichment Kit for Tissues and Cultured Cells (Thermo Scientific, 89839) according to the manufacturer’s instructions. Briefly, the DA neurons were pelleted and lysed with lysosome enrichment reagent A, which contained 1X protease and phosphatase inhibitors. The solution was then sonicated on ice, applying 12 bursts at 9W of power (Thermo Scientific) and mixed with lysosome enrichment reagent B. The solution was subsequently centrifuged at 500g for 10 minutes. 200 µL of supernatant was then aliquoted for use as cell supernatant for western blotting. Lysosomes were isolated from the remaining solution through gradient centrifugation. The protein concentrations of the lysosomes and cell supernatant were then quantified using a BCA assay and proceeded for western blotting as discussed below. SDS-PAGE and immunoblotting Fibroblasts and DA neurons were washed with ice-cold PBS, followed by the addition of 150 µl of ice-cold RIPA lysis buffer (Sigma-Aldrich, R0278), which was supplemented with protease inhibitors (Roche 4693116001), PMSF (Sigma, 93482), and phosphatase inhibitors (Roche 4906837001). Cells were scraped using a plastic scraper, and the lysates were transferred to 1.5 ml tubes. The lysates were rotated at 4°C for 30 minutes and sonicated (3 cycles, 3 seconds each at 40% amplitude, with 5-minute intervals). Samples were centrifuged at 16,000 g for 30 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA assay kit (Thermo Scientific, 23227). A total of 20–30 µg of protein was diluted with RIPA buffer and mixed with NuPAGE 4X sample buffer (Invitrogen, NP0007). The samples were heated at 95°C for 5 minutes (for OXPHOS proteins, the lysates were heated at 45ºC for 5 minutes). Proteins were separated on 4–12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen, NP0335) immersed in MOPS running buffer (Invitrogen, NP0001). Proteins were transferred to PVDF membranes (Millipore, IPFL00010) activated in methanol using a wet transfer system. Membranes were blocked in Superblock blocking buffer (Invitrogen, 37545) for 1 hour at room temperature. The blots were cut where necessary before incubating with primary antibodies, which were diluted in 1X blocking buffer, and incubated with the membranes overnight at 4°C. After three 10-minute washes in TBST, the membranes were incubated with secondary antibodies (Li-COR Biosciences; 1:10000; IRDye® 680RD Goat anti-Mouse IgG, 926-68070; IRDye® 800CW Goat anti-Rabbit IgG, 926-32211) diluted in 1% BSA/TBST for 1 hour at room temperature. The membranes were washed three additional times with TBST. Fluorescent signals were developed using a LiCOR Odyssey CLx system. The details of the antibodies are listed in Table 2 . Table 2 List of antibodies used in the study Name Catalogue no. Host Company Application Concentration ATP6V0D2 AB194557 Mouse Abcam Western Blotting 1:1000 LAMP1 (H4A3) sc-20011 Mouse Santa Cruz Western Blotting, Immunofluorescence 1:500 GBA (c-term) 64171 Rabbit Sigma Western Blotting 1:700 ATP6V1H AB187706 Rabbit Abcam Western Blotting 1:1000 ATP6V1A 199326 Rabbit Abcam Western Blotting 1:1000 pMTOR 55365 Rabbit Abcam Western Blotting 1:1000 MTOR 45175 Mouse Cell Signalling Western Blotting 1:1000 ATG5 (D5F5U) 12994T Rabbit Cell Signalling Western Blotting 1:1000 P62 610833 Mouse BD Biosystems Western Blotting 1:1000 LC3 L7543 Rabbit Sigma Western Blotting 1:1000 B-ACTIN ab8226 Mouse Cell Signalling Western Blotting 1:5000 TOM20 ab186735 Rabbit Abcam Western Blotting 1:1000 LC3 PM036 Rabbit MBL Biosystems Immunofluorescence 1:500 Citrate Synthetase ab96600 Rabbit Abcam Immunofluorescence 1:200 OXPHOS Cocktail 45-8199 Mouse Thermoscientific Western Blotting 1:1000 Alexa Fluor 488 A-11008 Goat Thermoscientific Immunofluorescence 1:1000 Alexa Fluor 594 A-11012 Goat Thermoscientific Immunofluorescence 1:1000 Alexa Fluor 647 A-21235 Goat Thermoscientific Immunofluorescence 1:1000 IRDye® 680RD Goat anti-Mouse 926-68070 Goat Li-COR Biosciences Western Blotting 1:10000 IRDye® 800CW Goat anti-Rabbit IgG 926-32211 Goat Li-COR Biosciences Western Blotting 1:10000 Immunostaining Fibroblasts were grown on coverslips, treated, and fixed with 4% (w/v) paraformaldehyde. After fixation, the cells were permeabilised with 50 µg/ml digitonin in PBS for 10 minutes. The cells were then washed, blocked with 3% BSA, and incubated with the following primary antibodies: citrate synthase, LAMP1, and LC3 in 3% BSA for 1 hour at room temperature, followed by incubation with Alexa Fluor 488/594/647-conjugated secondary antibodies for 1 hour at room temperature. Coverslips were mounted on glass slides, and images were acquired using appropriate excitation and emission filters to capture fluorescent signals. Colocalisation Pearson’s coefficient (Mito vs LC3 and Mito vs LAMP1), lysosomal number (LAMP1 particles/cell), and autophagosome number (LC3 particles/cell) were quantified using ImageJ/Fiji with consistent threshold settings across all samples. The catalogue number and dilution range of the antibodies are listed in Table 2 . Mitochondrial isolation Mitochondria were isolated according to the method described earlier [ 74 ] and modified for the neuronal cultures. Briefly, DA neuron cell pellets were resuspended in mitochondria isolation buffer (MIB1: 225 mM Mannitol, 75 mM Sucrose, 5 mM HEPES, 1 mM EGTA and 1 mg/ml fatty acid free BSA) and homogenised using a ice-cold Dounce tissue grinder tube (appropriate for 1-1.5 ml homogenisation volume). The homogenate was centrifuged, and the mitochondrial pellet was washed and pellet down in MIB without BSA (MIB2) according to the protocol. The final pellet was resuspended in a very small volume of MIB2, and the protein concentration was determined using BCA Protein assay kit (Thermofisher Scientific, 23227) according to the manufacturer’s specifications.The isolated mitochondria were then used for respiratory measurements. Measurement of oxygen consumption rate Measurements of mitochondrial respiration were conducted with the Seahorse Bioscience XFe96 bioanalyzer using the Seahorse XF Cell Mito Stress Test Kit (Agilent #103015-100). Final maturation of iPSCs into DA neurons were performed in XF96 cell culture microplates (Agilent #102416-100). On the day of the experiment, the culture medium was replaced with Seahorse XF Base medium (Agilent #103334-100) supplemented with 1 mM pyruvate (Gibco #11360070), 2 mM glutamine (Gibco #25030081) and 10 mM glucose (Gibco #A2494001) and incubated for 30 min at 37°C in a CO 2 -free incubator before loading into the Seahorse Analyser. After measuring basal respiration, the drugs oligomycin (5 µM), FCCP (1.5 µM), and rotenone/antimycin A (0.5 µM/0.5 µM) were added to each well in sequential order. After the assay, cells were stained with Hoechst 33342 (5 µM; Thermo Scientific #62249) for 30 min. ImageXpress was then used to count the number of cell nuclei (cell numbers) in each well and normalised to get the basal respiration rate values. The respiratory measurements of isolated mitochondria from mixed neuronal cultures were performed using a modified method described earlier[ 75 ] for Seahorse XFe96 cell culture plates assay. Briefly, 5 or 10 µg of mitochondrial protein was resuspended in 30 µL (1 well) of individual substrate mix (for example, pyruvate/malate substrate + mitochondria assay solution - MAS) and plated into each well. The cell culture plate was centrifuged at 2,000g for 20 minutes at 4°C to form a uniform layer of mitochondria at the bottom. After centrifugation, 150 µL of substrate solution was carefully added to each well. Fresh injection solutions were made in MAS without BSA and loaded into the cartridge, and calibrated according to the manufacturer’s specifications. After calibration, the culture plate with mitochondria was inserted, and the assay was run essentially as described in the method. Transmission electron microscopy (TEM) TEM was performed as previously described [ 72 ]. Briefly, fibroblasts and DA neurons grown on coverslips were fixed in electron microscopy (EM) fixative containing 2% glutaraldehyde (EMS, 16365) and 2% paraformaldehyde (EMS, 15710) in 0.1 M sodium cacodylate for 1 hour. Following fixation, cells were washed with 0.1 M cacodylate buffer (EMS, 11650) and then fixed in a solution of 1% osmium tetroxide (EMS, 19150) and 1% potassium ferricyanide (EMS, 25120-20) in 0.1 M sodium cacodylate. This was followed by sequential dehydration using ethanol. Coverslips were then embedded in epoxy resin (Araldite Kit, Agar Scientific Ltd., CY212) according to standard protocols. The embedded samples were sectioned into 50 nm slices using an ultramicrotome equipped with a diamond knife and mounted onto copper grids suitable for TEM. The grids were stained with lead citrate for 3 minutes before imaging. Images were captured using a Jeol 1400 Transmission Electron Microscope at magnifications ranging from 800× to 1200× (digital magnification). Images were imported into ImageJ/Fiji to quantify mitochondrial area, mitochondrial cristae density, and the number of autophagosomes, autolysosomes, and lysosomes per field. Mitochondria and autophagic vesicles were manually traced to determine the area. Quantification and statistical analysis All statistical analyses were performed using GraphPad Prism. For comparisons between two groups, a two-tailed unpaired t-test was applied to normally distributed data. Multi-group comparisons were analysed using one-way ANOVA followed by Tukey’s multiple comparisons test or two-way ANOVA. Data are presented as mean ± SEM. from at least three independent replicates, with 50–70 cells per repeat for DA neurons and 10–15 cells per repeat for fibroblasts for live cell imaging experiments. For immunoblotting or TMRM data, control sample means were normalised to one to facilitate comparisons. Statistical significance was defined as p < 0.05. p-values are reported as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001, ns – not significant. Abbreviations GBA1 Glucocerebrocidase PD Parkinson’s Disease GCase β-glucocerebrosidase enzyme pH potential of Hydrogen V ATPase -Vascular ATPase iPSCs induced Pluripotent Stem Cells DA neuron Dopaminergic neurons OXPHOS Oxidative Phosphorylation ΔΨm Mitochondrial Membrane Potential TMRM TetraMethyl-Rhodamine Methyl ester MTORC1 Mammalian Target of Rapamycin Complex I FLIM FRET -Fluorescence Lifetime Imaging (FLIM) with Förster Resonance Energy Transfer NPs Nanoparticles Declarations Ethics approval The source and ethical approval committee for the iPSCs used in the study is tabulated in table 1. Consent for publication Not applicable Competing interests The authors declared no competing interests in this research. Funding This project was funded by the Michael J Fox Foundation (Project number E27234 to MRD) and the Parkinson’s UK Foundation (G-2103 to MRD and PS). Authors' contributions P.S., M.R.D - Conceptualisation, funding acquisition, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. A.C.B –methodology, investigation, visualisation and data analysis. S.K, A.F, I.K, K.S and T.S.B– Methodology, investigation and data analysis. O.S, J.Z and M.G – Resources. Acknowledgements The authors would also like to express their gratitude to the members of the Michael Duchen and Gyorgy Szabadkai labs for their feedback, as well as to students, including Shail Bhatt and Oriane Marguet, for their assistance with the preliminary data. 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GCase activity in GBA1-E326K (Bi a) and GBA1-N370S (Bi b) DA neurons and respective isogenic controls. Representative ratioed images (Ci a, b) and quantification of fluorescence ratio of GBA1-E326K (Cii a) and GBA1-N370S (Cii b) DA neurons and respective isogenic controls labelled with Lysosensor Yellow/Blue DND 160 indicating lysosomal pH in cells. D) Histogram depicting mean lysosomal pH values in \u003cem\u003eGBA1 \u003c/em\u003emutant fibroblasts. Representative confocal images (Ei a,b) and quantification of relative intensity of GBA1-E326K (Eii a) and GBA1-N370S (Eii b) DA neurons and respective isogenic controls stained with DQ-Red BSA indicative of proteolytic activity. Representative confocal images (Fi) and quantification (Fii) of lipofuscins which are seen upon autofluorescence. Data represented as mean±SEM; n=3; Statistics:**** - p\u0026lt;0.001; *** - p\u0026lt;0.005; ** - p\u0026lt;0.01, * - p\u0026lt;0.05; ns – not significant; Mann-Whitney’s T test, One-way ANOVA.\u003c/p\u003e","description":"","filename":"Figurescombined1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/31a0e2227507e13436b52696.jpg"},{"id":93006274,"identity":"3a7749a0-f71f-455d-b7d1-a8cb6288d9b3","added_by":"auto","created_at":"2025-10-08 06:46:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2721777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial dysfunction in GBA1-PD DA neurons: \u003c/strong\u003eRepresentative confocal images of GBA1-E326K (Ai a) and GBA1-N370S (Ai b) DA neurons along with isogenic controls stained with TMRM and subsequent quantification of fluorescent intensity to measure DΨm (Aii a, b) and mitochondrial fragmentation count (Aii c,d). Representative ultramicroscopic images of isogenic control and GBA1-E326K (Bi a) and GBA1-N370S (Bi b) DA neurons and subsequent quantification of morphological parameters including average number of mitochondria, average mitochondrial area and average cristae density in mitochondria (Bii a-f). The white arrow indicates normal mitochondria; black arrow indicates abnormal mitochondria. Histograms representing the basal respiration rate in \u003cem\u003eGBA1\u003c/em\u003e mutant iPSC-DA neurons from GBA1-E326K (C a) and GBA1-N370S mutant lines(C b) and respective isogenic controls. Representative seahorse plots showing oxygen consumption rates in iPSC-DA neurons from GBA1-E326K (Di a) and GBA1-N370S (Di b) DA neurons and respective isogenic controls, using various substrates. Histograms representing State 3(ATP Production) in mitochondria isolated from GBA1-E326K (Dii a) and GBA1-N370S (Dii b) DA neurons and isogenic controls, as measured by seahorse assay. E i and ii) Representative immunoblotting images (Ei) with subsequent quantification of OXPHOS complex proteins in GBA1 E326K (Eii a) and N370S (Eii b) DA neurons. Data represented as mean±SEM; n=3; Statistics:**** - p\u0026lt;0.001; *** - p\u0026lt;0.005; ** - p\u0026lt;0.01, * - p\u0026lt;0.05; ns – not significant; Mann-Whitney’s T-test, One-Way ANOVA or Two-Way ANOVA.\u003c/p\u003e","description":"","filename":"Figurescombined2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/2a50a1ee0c90d9579067fd55.jpg"},{"id":93006272,"identity":"997f0c2c-d5f2-49bf-8458-afba3a8b4223","added_by":"auto","created_at":"2025-10-08 06:46:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2481320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitophagy defects in GBA1-PD DA neurons: \u003c/strong\u003eRepresentative images of GBA1-E326K (Ai a)and GBA1-N370S (Ai b) DA neurons and isogenic controls transduced with mt-Keima plasmid and imaged with excitation at 458 and 543nm and subsequent quantification of the ratio of signals at 543/458nm (Aii a,b). Representative western blot images of \u003cem\u003eGBA1\u003c/em\u003emutant and isogenic control lysates probed for autophagic proteins (Bi) and subsequent quantification of protein levels (Bii a,b) Representative Immunofluorescence images of Control and N370S fibroblasts probed against Citrate Synthase (CiS), LAMP1 and LC3, labelling mitochondria, lysosomes and autophagosomes respectively (Ci) and subsequent quantification of colocalisation co-efficient of mitophagosomes (Cii a) and mitolysosomes(Cii b). Representative TEM images (Di) and quantification (Dii) of control and E326K mutant fibroblasts capture different autophagy phases in the cells. Black arrows indicate lipofuscins, yellow arrows indicate autolysosomes, red arrows indicate autophagosomes, and green arrows indicate lysosomes in the cells. Data represented as mean±SEM; n=3; Statistics:**** - p\u0026lt;0.001; *** - p\u0026lt;0.005; ** - p\u0026lt;0.01, * - p\u0026lt;0.05; ns – not significant; Mann-Whitney’s T-test, One-Way ANOVA.\u003c/p\u003e","description":"","filename":"Figurescombined3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/8338f192aa02ef2efb41b95d.jpg"},{"id":93006269,"identity":"dd23a9a8-c337-4698-a3f9-7fbe5ff7b1d4","added_by":"auto","created_at":"2025-10-08 06:46:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1940744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired V-ATPase complex formation in GBA1-PD cells- \u003c/strong\u003eRepresentative images of western blots (Ai) and quantification of protein levels in total cell lysates from isogenic control and GBA1-E326K (Aii a) and GBA1-N370S (Aii b) mutant DA neurons probed against LAMP1 and V-ATPase complex proteins. Representative immunoblotting images of cell supernatant and lysosomes separated by lysosome enrichment assay in untreated (Bi a) and 200nM rapamycin-treated (Bi b) GBA1-PD DA neurons and isogenic controls, and subsequent quantification (Bii) of V-ATPase complex proteins, pMTOR. Representative FLIM images of ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeonGreen in control and GBA1-PD fibroblasts co-transfected with ATP6V\u003csub\u003e0\u003c/sub\u003ea3-mScarlet and treated with/without 200nM rapamycin (Ci) and subsequent quantification of mean lifetime (τ\u003csub\u003em\u003c/sub\u003e) (Cii b). Cii a) Representative decay plots and exponential fit of ATP6V1B2-mNeongreen in the Control and N370S and E326K mutant fibroblasts. D i) Time point images of ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeongreen in control and GBA1-PD fibroblasts co-transfected with ATP6V\u003csub\u003e0\u003c/sub\u003ea3-mScarlet and treated with 1 µM rapamycin and imaged over a period of 40 mins and subsequent quantification of τ\u003csub\u003em \u003c/sub\u003eshown in Dii)\u0026nbsp; Data represented as mean±SEM; n=3; Statistics:**** - p\u0026lt;0.001; *** - p\u0026lt;0.005; ** - p\u0026lt;0.01, * - p\u0026lt;0.05; ns – not significant; Mann-Whitney’s T-test, One-Way ANOVA or Two-Way ANOVA.\u003c/p\u003e","description":"","filename":"Figurescombined4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/8911193b1542399b4c6d2a7f.jpg"},{"id":93006270,"identity":"a667ee8e-3e38-421e-be0f-aecbdda4edfe","added_by":"auto","created_at":"2025-10-08 06:46:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2100196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapamycin and acidic NPs rescue lysosomal and mitochondrial dysfunction in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGBA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant DA neurons: \u003c/strong\u003eRepresentative ratioed images of GBA1-E326K (Ai a) and GBA1-N370S (Ai b) DA neurons and respective isogenic controls treated overnight with 200nM rapamycin or 180mg/mL acidic NPs and stained with Lysosensor Yellow/Blue DND 160 (Ai a,b) and quantification of fluorescence ratio (Aii a-d) indicating lysosomal pH. Representative confocal images of GBA1-E326K (Bi a) and GBA1-N370S (Bi b) DA neurons and respective isogenic controls treated with 200nM rapamycin or 180mg/mL acidic NPs and stained with TMRM to measure changes in DΨm (Bii a-d). Representative confocal Images of GBA1-E326K (Ci a) and GBA1-N370S (Ci b) DA neurons and respective isogenic controls treated with 200nM rapamycin or 180mg/mL acidic NPs and probed with mt-Keima plasmid and imaged with excitation at 458 and 543nm and subsequent quantification of the ratio of signals at 543/458nm (Cii a-d). Data represented as mean±SEM; n=3; Statistics:**** - p\u0026lt;0.001; *** - p\u0026lt;0.005; ** - p\u0026lt;0.01, * - p\u0026lt;0.05; ns – not significant; One-Way ANOVA.\u003c/p\u003e","description":"","filename":"Figurescombined5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/119d11106ea0b81cdd764032.jpg"},{"id":93007653,"identity":"33013299-fa96-4bea-92a0-51b3e9027520","added_by":"auto","created_at":"2025-10-08 06:54:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":663406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCartoon to illustrate the proposed pathophysiological cascade in GBA1-related Parkinson’s Disease and how the phenotype can be corrected by targeting lysosomal pH - \u003c/strong\u003eGBA1-PD cells exhibit a range of abnormalities in lysosomal biology, including reduced GCase activity, increased lysosomal numbers, elevated lysosomal pH, and decreased pH-dependent proteolytic activity. Mitochondrial dysfunction is evidenced by a decreased DΨm, decreased oxygen consumption rate, swollen mitochondria, reduced cristae density, and the accumulation of abnormal mitochondria. Mitophagy is suppressed and autolysosomes accumulate within the GBA1-PD cells. Restored acidification of lysosomes with rapamycin or acidic poly-succinate NPs enhances lysosomal function and improves mitophagy, thereby rescuing mitochondrial dysfunction in GBA1-PD fibroblasts and lysosomes.\u003c/p\u003e","description":"","filename":"Figurescombined6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/6e497f7ba1587f50d0f9122a.jpg"},{"id":93007937,"identity":"a65f62e7-102b-4bf4-b232-e98dc8f200dc","added_by":"auto","created_at":"2025-10-08 07:02:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12917879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/a2f23a29-2a1d-49ab-8d86-1702f362c791.pdf"},{"id":93006280,"identity":"d7bfd2fc-a4c3-4ba8-abaa-6d0aa1041ed8","added_by":"auto","created_at":"2025-10-08 06:46:42","extension":"pptx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":148919017,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalBlots.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/50810eb20e4c47611842d32c.pptx"},{"id":93006275,"identity":"3154decc-af08-4607-8215-23a66f95795c","added_by":"auto","created_at":"2025-10-08 06:46:40","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":922243,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureswithlegend.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7558589/v1/894fbcc15cdc4600e7f782ab.pdf"}],"financialInterests":"","formattedTitle":"Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinson’s Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenome-wide association studies have revealed that mutations of the glucocerebrosidase (\u003cem\u003eGBA1\u003c/em\u003e) gene constitute a significant risk factor in the development of Parkinson\u0026rsquo;s Disease (PD)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The \u003cem\u003eGBA1\u003c/em\u003e gene encodes the enzyme β-glucocerebrosidase (GCase), which generates glucose and ceramide from glucosylceramide within lysosomes. While homozygous \u003cem\u003eGBA1\u003c/em\u003e mutations cause Gaucher Disease, which may include a significant neurodegenerative component[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], heterozygous mutations are associated with an increased risk of developing PD. The two most prevalent \u003cem\u003eGBA1\u003c/em\u003e mutations associated with PD are the N370S and L444P mutations[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Intriguingly, the less common E326K mutation in \u003cem\u003eGBA1\u003c/em\u003e exhibits a relatively mild effect on GCase activity, does not cause Gaucher\u0026rsquo;s Disease, but is correlated with PD risk[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMitochondrial dysfunction appears to constitute a defining characteristic of PD[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The adverse impact of dysfunctional mitochondrial oxidative phosphorylation (OXPHOS) on the viability of dopaminergic (DA) neurons is substantiated by experimental models of PD that result from toxins that target complex I[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The \u003cem\u003eGba1\u003c/em\u003e mouse knockout model, a model of severe neurodegeneration, exhibited severe mitochondrial dysfunction in primary neurons and astrocytes in culture, alongside neurological pathologies associated with PD, including disruption of autophagy-lysosomal pathways, and the accumulation of ubiquitinated proteins and α-synuclein[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe autophagosome-lysosome axis and ubiquitin proteasome systems accomplish degradation of dysfunctional organelles and protein degradation and together play a critical role in cellular quality control[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Lysosomal acidification generating pH values between 4.5 to 4.7[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] is essential for normal lysosomal function and is required for the activity of lysosomal hydrolytic enzymes and effective protein degradation[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The acidification is generated by the vacuolar-type H\u003csup\u003e+\u003c/sup\u003e ATPase (V-ATPase), a proton pump composed of a peripheral V\u003csub\u003e1\u003c/sub\u003e domain, which hydrolyses ATP, and a membrane-integrated V\u003csub\u003eo\u003c/sub\u003e domain, responsible for the translocation of protons into the lysosomal lumen[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Disrupted V-ATPase assembly and V-ATPase dysfunction have been reported associated with multiple disorders[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], including Juvenile-onset Parkinson\u0026rsquo;s Disease[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Alzheimer\u0026rsquo;s Disease[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Epilepsy[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and Down\u0026rsquo;s syndrome[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eGBA1\u003c/em\u003e-linked pathologies such as Gaucher\u0026rsquo;s disease and PD are associated with a dysfunctional autophagy-lysosome pathway, including impaired lysosomal regeneration from autolysosomes during macroautophagy[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These investigations suggest that targeting the dysfunctional lysosomal system and the autophagy-lysosome pathway may serve as a potential therapeutic strategy for PD linked to \u003cem\u003eGBA1\u003c/em\u003e mutations. Although numerous studies indicate that impaired lysosomal acidification plays a role in neuronal pathologies associated with various neurodegenerative disorders[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and that GBA1-PD is characterised by lysosomal dysfunction [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], the effects of compromised lysosomal acidification in GBA1-PD have not been extensively examined.\u003c/p\u003e\u003cp\u003eIn this study, we have characterised the consequences of \u003cem\u003eGBA1\u003c/em\u003e mutations associated with PD for mitochondrial and lysosomal function in patient-derived fibroblasts and DA neurons generated from patient-derived induced pluripotent stem cells (iPSC). As GBA1 is a lysosomal enzyme, we hypothesised that lysosomal dysfunction as a consequence of the mutations, might lead to impaired mitochondrial function, initiating a pathophysiological cascade culminating in cell injury. Furthermore, we found that constitutive and inappropriate activation of mechanistic target of rapamycin complex 1 (MTORC1) contributes to impaired lysosomal acidification through aberrant assembly of the V-ATPase complex, leading to lysosomal dysfunction. Additionally, we demonstrate that the restoration of lysosomal pH ameliorates autophagy and rescues both lysosomal and mitochondrial function in cells carrying GBA1-PD mutations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo characterise the impact of PD-related \u003cem\u003eGBA1\u003c/em\u003e mutations on lysosomal and mitochondrial function, we examined three lines of human dermal fibroblasts and six iPSC lines derived from GBA1-PD patients carrying E326K or N370S mutations, along with two healthy controls. We also included one CRISPR-corrected isogenic control iPSC line each for GBA1-N370S and GBA1-E326K iPSCs. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; List of cell lines used\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell Lines used\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell Line ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAge/Sex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCell type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMutation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eEthics Committee Approval\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCONTROL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibroblasts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo Mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNHNN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN370S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eND34263\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibroblasts; source for NH50182 iPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-N370S; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCONTROL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibroblasts; source for Control1 iPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo Mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNHNN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE326K 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHet1/ JS48753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibroblasts; source for Het1/E326K 1 iPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-E326K; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNHNN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE326K 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eND41015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibroblasts; source for ND50045 iPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-E326K; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsogenic Control (IsoC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNH50142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs; Isogenic Control for ND50045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCRISPR corrected GBA1-E326K heterozygous mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE326K\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eND50045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-E326K; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsogenic Control (IsoC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNH50186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs; Isogenic Control for NH50182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCRISPR corrected GBA1-N370S heterozygous mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN370S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNH50182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-N370S; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNIH-RUCDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo Mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDerived from CONTROL 2 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics for CONTROL 2 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE326K 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHet1/ JS48753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-E326K; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDerived from Het1 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics for JS48753 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE326K 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHet 2/ MB240649\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-E326K; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDerived from MB240649 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePD Royal Free Ethics (for MB240649 fibroblasts\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSFC156 (Control 2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo Mutation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEBiSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN370S 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSFC834 (N370S1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e72/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-N370S; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEBiSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN370S 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSFC848 (N370S2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68/Male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eiPSCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGBA1-N370S; Heterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEBiSC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eLysosomal function is impaired in cells with PD-related GBA1 mutations\u003c/h2\u003e\u003cp\u003eAs GBA1 is a lysosomal enzyme, our investigation began by characterising the lysosomes in fibroblasts and DA neurons from both control and PD patient-derived iPSCs. The GBA1 protein expression levels measured by western blot were not significantly different between the control and \u003cem\u003eGBA1\u003c/em\u003e mutant neurons; however, GCase enzymatic activity was significantly reduced in iPSC-DA neurons and fibroblasts carrying either \u003cem\u003eGBA1\u003c/em\u003e mutation (Fig.\u0026nbsp;1A, B, S1A, B). Lysosomal acidification was measured using the ratiometric pH-sensitive dye, Lysosensor Yellow/Blue DND160. These data revealed impaired acidification in both E326K and N370S DA neurons and fibroblasts (Fig.\u0026nbsp;1C, S1C, D). Calibration of the lysosomal pH (see methods) gave values of 4.74\u0026plusmn;0.03\u0026ndash;4.77\u0026plusmn;0.02 in control cells but between 4.89\u0026plusmn;0.02 and 5.06\u0026plusmn;0.03 in the mutant fibroblasts (Fig.\u0026nbsp;1D). The function of endocytic trafficking and lysosomal proteolytic activity was measured using the DQ-Red BSA assay. DQ-Red BSA intensity was significantly reduced in the \u003cem\u003eGBA1\u003c/em\u003e mutant neurons, indicating impaired lysosomal degradative function in GBA1-PD cells (Fig.\u0026nbsp;1E, S1E). Autofluorescence excited at 355nm and imaged between 400-600nm showed an unusually bright extramitochondrial component in GBA1-PD fibroblasts (Fig.\u0026nbsp;1F) that was significantly greater than control cells. Spectral scanning and linear unmixing separated the expected mitochondrial NADH signal with an emission peak at 450nm and an extensive non-mitochondrial signal with a peak emission at 480nm, attributed to accumulated lipofuscin. Lipofuscins are cytoplasmic granules generated as a consequence of autophagy and phagocytosis processes, which show auto-fluorescence over a broad spectrum ranging from 480nm to 700nm when excited by ultraviolet or blue light[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While the accumulation of lipofuscin serves as an indicator of ageing, abnormal accumulation is consistent with impaired lysosomal acidification and defective autophagy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Accumulation of lipofuscin is also a hallmark of Batten Disease (neuronal ceroid-lipofuscinoses), a severe early-onset neurodevelopmental disorder with progressive neurodegeneration also associated with lysosomal dysfunction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These data collectively indicate significant lysosomal dysfunction in the GBA1-PD cells.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMitochondria are dysfunctional in cells with GBA1-PD mutations\u003c/h3\u003e\n\u003cp\u003eTo determine whether the impaired lysosomal activity in the \u003cem\u003eGBA1\u003c/em\u003e mutations has a secondary impact on mitochondrial form and function, we measured mitochondrial membrane potential (ΔΨm) using the potentiometric fluorescent reporter tetramethyl-rhodamine methyl ester (TMRM). ΔΨm was significantly reduced in E326K and N370S \u003cem\u003eGBA1\u003c/em\u003e mutant DA neurons and fibroblasts (Fig.\u0026nbsp;2A, S2A, C). The lower ΔΨm was accompanied by mitochondrial fragmentation in the N370S and E326K DA neurons and fibroblasts (Fig.\u0026nbsp;2Aii c,d, S2Aii b). Ultrastructure analysis using electron microscopy revealed significant differences in mitochondrial morphology in the \u003cem\u003eGBA1\u003c/em\u003e mutant neurons, including reduced cristae density and increased mitochondrial area, indicating mitochondrial swelling in the \u003cem\u003eGBA1\u003c/em\u003e mutant iPSC DA neurons (Fig.\u0026nbsp;2B). Measurements of oxygen consumption rates using the \u0026lsquo;Seahorse\u0026rsquo; respirometry system revealed\u0026thinsp;~\u0026thinsp;40% decrease in basal oxygen consumption rate in the \u003cem\u003eGBA1\u003c/em\u003e mutant DA neurons with the isogenic controls (Fig.\u0026nbsp;2C). To exclude changes in mitochondrial mass and to explore the substrate dependence of mitochondrial respiration, we used mitochondria isolated from the cultures. This allowed measurements of respiratory rates with different substrates, favouring complex I (pyruvate/malate) or complex II (rotenone/succinate). ADP-stimulated respiration (State 3) rate using both CI and CII-dependent substrates was significantly reduced in the \u003cem\u003eGBA1\u003c/em\u003e mutant neurons compared to the control neurons (Fig.\u0026nbsp;2D, S2C). Western Blots of the OXPHOS complex proteins using a cocktail of antibodies to respiratory chain proteins, revealed a significant increase in the expression of Complex IV in E326K DA neurons (Fig.\u0026nbsp;2Eii a and S2Dii a) and increased expression of Complex I proteins in N370S neurons compared to the isogenic controls (Fig.\u0026nbsp;2Eii b and S2Dii b). Expression levels of the other respiratory chain proteins were unaltered in the E326K and N370S DA neurons. None of these data suggested any defect in complex I assembly or function. These data collectively point to impaired mitochondrial bioenergetic function in fibroblasts and neurons carrying \u003cem\u003eGBA1\u003c/em\u003e mutations.\u003c/p\u003e\n\u003ch3\u003eMitophagy is impaired in cells with GBA1-PD mutations\u003c/h3\u003e\n\u003cp\u003eWe then set out to explore the underlying mechanisms that link impaired lysosomal function to impaired mitochondrial bioenergetics in the GBA1-PD fibroblasts and DA neurons. A logical mechanism linking impaired lysosomal function with mitochondrial dysfunction might operate through dysfunctional mitophagy and our earlier work also established dysfunctional mitophagy in \u003cem\u003eGba1\u003c/em\u003e KO mice[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We quantified mitophagy using the dual excitation probe mt-Keima (Fig.\u0026nbsp;3A). mt-Keima is a pH-sensitive probe that measures the fraction of mitochondria in neutral (pH 7\u0026ndash;7.8 in the mitochondrial matrix) versus acidic pH (pH 4.5\u0026ndash;4.7 in lysosomes) environment[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The mt-Keima signal ratio was significantly reduced in fibroblasts and DA neurons in the fibroblasts and DA neurons carrying both the GBA1-N370S and GBA1-E326K mutations indicating impaired mitophagy (Fig.\u0026nbsp;3Aii. S3Aii, Bii). Western blotting to quantify the status of autophagy pathways in the \u003cem\u003eGBA1\u003c/em\u003e mutant neurons revealed increased LAMP1 levels in E326K and N370S neurons, along with increased MTORC1 phosphorylation in the two mutant types. While LC3 flux, and expression of p62 and TOM20 were increased in the E326K mutant iPSC DA neurons, these were not significantly altered in the N370S DA neurons (Fig.\u0026nbsp;3B). Since the lysosomal pH is increased in GBA1-PD cells (Fig.\u0026nbsp;1D), the reduced mt-Keima signal in GBA1-PD cells could be attributed either to the altered lysosomal pH or to the impaired fusion of autophagosomes to lysosomes. To address this, we performed triple staining of fibroblasts against citrate synthase (CiS, for mitochondria), LAMP1 (for lysosomes), and LC3 (for autophagosomes). Subsequent imaging revealed that although there was no significant difference in mitophagosome (mitochondria and autophagosome colocalisation) density between \u003cem\u003eGBA1\u003c/em\u003e mutant and control fibroblasts, mitolysosome (colocalisation of mitochondria and lysosomes) density was significantly increased in the \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts (Fig.\u0026nbsp;3C). Characterisation of electron micrographs for specific autophagic vesicle types based on Neikirk et al.,2023[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], showed that autophagic vesicles, specifically the autolysosome and lysosome number, were significantly increased in both \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts and neurons. (Fig.\u0026nbsp;3Dii, S3Cii a and b). From the micrographs, the autophagosomes also appeared to be fully enclosed, ruling out incomplete phagophore formation as the underlying mechanism of autophagy defect. These data indicated that mitophagy is significantly impaired in cells carrying the GBA1-PD mutations and the decrease in mitophagy is likely due to impaired pH and not improper phagophore formation.\u003c/p\u003e\n\u003ch3\u003eV-ATPase complex formation is impaired in GBA1-PD\u003c/h3\u003e\n\u003cp\u003eSince our data showed an accumulation of lysosomes and autolysosomes in GBA1-PD fibroblasts and iPSC-DA neurons, we suspected this was a result of impaired lysosomal acidification. Assembly of the pH regulatory component in lysosomes \u0026ndash; the Vacuolar-type H\u0026thinsp;+\u0026thinsp;ATPase (V-ATPase) is regulated by MTORC1[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which was constitutively phosphorylated in the GBA1-PD mutant cells. Ratto et al.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] demonstrated that under nutrient-rich conditions, MTORC1 is distributed in the cytosol, enabling the peripheral ATP6V\u003csub\u003e1\u003c/sub\u003e to bind to the membrane-bound ATP6V\u003csub\u003e0\u003c/sub\u003e domain, forming a functional V-ATPase complex allowing proton exchange and acidification of the lysosomes.\u003c/p\u003e\u003cp\u003eUpon nutrient deprivation, MTORC1 is phosphorylated and remains on the lysosomal membrane, preventing the formation of a functional V-ATPase complex[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While the V-ATPase maintains lysosomal pH, disruption of the complex does not hamper autolysosome formation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Total protein estimation through western blotting revealed no significant difference in the expression of ATP6V\u003csub\u003e0\u003c/sub\u003eD2 and ATP6V\u003csub\u003e1\u003c/sub\u003eA or ATP6V\u003csub\u003e1\u003c/sub\u003eH between the control and mutant DA neurons (Fig.\u0026nbsp;4A). To examine their expression levels in lysosomes specifically, we performed a lysosomal enrichment assay and probed for pMTORC1, LAMP1, and the ATP6V\u003csub\u003e0\u003c/sub\u003e and ATP6V\u003csub\u003e1\u003c/sub\u003e components (Fig.\u0026nbsp;4Bi). pMTORC1 expression was increased up to two-fold, and LAMP1 expression was increased\u0026thinsp;~\u0026thinsp;1.5 fold in the GBA1-mutant DA neurons. However, ATP6V\u003csub\u003e1\u003c/sub\u003eA expression was reduced by ~\u0026thinsp;40% and ATP6V\u003csub\u003e1\u003c/sub\u003eH by ~\u0026thinsp;15% within the lysosomes fractionated from \u003cem\u003eGBA1\u003c/em\u003e mutant DA neurons, while ATP6V\u003csub\u003e0\u003c/sub\u003eD2 levels remained unchanged between the control and mutant iPSC-DA neurons (Fig.\u0026nbsp;4Bii). Furthermore, overnight treatment of \u003cem\u003eGBA1\u003c/em\u003e iPSC-DA neurons with the MTORC1 inhibitor rapamycin (200nM) decreased the pMTORC1 and LAMP1 levels, while increasing ATP6V\u003csub\u003e1\u003c/sub\u003eA and ATP6V\u003csub\u003e1\u003c/sub\u003eH expression in the lysosomes and the ATP6V\u003csub\u003e0\u003c/sub\u003eD2 levels remained constant (Fig.\u0026nbsp;4Bi b and Bii).\u003c/p\u003e\u003cp\u003eIn order to assess the V-ATPase assembly, we transfected the cells with ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeongreen and ATP6V\u003csub\u003e0\u003c/sub\u003ea3-mScarlet and quantified their FRET interaction using fluorescence lifetime imaging microscopy (FLIM)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The data were best fit to a biexponential decay function. The ATP6V\u003csub\u003e0\u003c/sub\u003ea3 construct utilises the more rapidly maturing but photophysically heterogeneous acceptor variant mScarlet-I[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This exhibits at least two fluorescence lifetimes, meaning the biexponential decay of the donor is an oversimplification given the likelihood of contrasting FRET rates to each acceptor species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The results are therefore presented as the mean (amplitude weighted) fluorescence lifetime -τ\u003csub\u003em\u003c/sub\u003e- which will nevertheless respond to variations in FRET without having to interpret the meaning of individual decay components and amplitudes. t\u003csub\u003em\u003c/sub\u003e of ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeongreen averaged 617.72\u0026plusmn; 25.3 ps in control fibroblasts but increased to 760.88\u0026plusmn; 20.36 ps in N370S and 811.03\u0026plusmn; 17.04 ps in E326K mutant fibroblasts, indicating an increased distance between ATP6V1B2-mNeongreen and ATP6V0a3-mScarlet signalled by a reduction in FRET. Overnight treatment with 200nM rapamycin significantly decreased t\u003csub\u003em\u003c/sub\u003e to 652.7\u0026plusmn; 19.69 ps and 726.05\u0026plusmn; 21.2 ps in E326K and N370S fibroblasts respectively while the t\u003csub\u003em\u003c/sub\u003e control fibroblasts was at 590.5\u0026plusmn; 32.7 ps (Fig.\u0026nbsp;4C). To validate the subcellular localisation process within a condensed timeframe, we administered 1 \u0026micro;M rapamycin to the cells and performed FLIM every 10 minutes for 40 minutes. The mean fluorescence lifetime of ATP6V1B2-mNeongreen exhibited a progressive decline in the \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts, decreasing from ~\u0026thinsp;850 ps at 0 min to ~\u0026thinsp;580 ps at 40 minutes, while t\u003csub\u003em\u003c/sub\u003e in control fibroblasts showed only a small reduction, decreasing from ~\u0026thinsp;675 ps at 0 min to ~\u0026thinsp;588 ps at 40 minutes (Fig.\u0026nbsp;4Dii). The FLIM-FRET data thus confirmed impaired assembly of the V -ATPase complex in \u003cem\u003eGBA1\u003c/em\u003e mutant cells.\u003c/p\u003e\u003cp\u003eAll these data are consistent with a model in which phosphorylation of MTORC1 at the lysosome membrane limits the formation of a functional V-ATPase complex in the GBA1-PD cells. Employing rapamycin as a potent MTORC1 inhibitor reduced MTORC1 activity and facilitated the formation of a functional V-ATPase complex.\u003c/p\u003e\n\u003ch3\u003eAcidification of lysosomes is sufficient to restore lysosomal, mitochondrial function and mitophagy in GBA1-PD\u003c/h3\u003e\n\u003cp\u003eOur data show that \u003cem\u003eGBA1\u003c/em\u003e mutations lead to significantly impaired lysosomal acidification and mitophagy and suggest that this failure of cellular homeostasis may underlie the mitochondrial dysfunction seen in GBA1-PD cells. We therefore wondered whether restoring lysosomal pH could rescue these defects in GBA1-PD cells. Since impaired V-ATPase complex formation may be a consequence of MTORC1 hyperphosphorylation, we explored the impact of treatment with rapamycin (overnight treatment with 200 nM) on lysosomal and mitochondrial function. As an independent pH modulator, we employed novel poly(ethylene tetrafluorosuccinate-co-succinate) nanoparticles (NPs), which acidify lysosomes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe first validated the effects of rapamycin and the nanoparticles in patient-derived fibroblasts (Fig S4). To confirm that the NPs were localised to the lysosomes, we loaded the fibroblasts with rhodamine-tagged NPs and stained the cells with Lysotracker blue DND22 (Fig S4A) which confirmed the localisation of the NPs to lysosomes. Overnight treatment with 180 \u0026micro;g/mL acidic NPs composed of poly(ethylene tetrafluorosuccinate-co-succinate) restored lysosomal pH in \u003cem\u003eGBA1\u003c/em\u003e mutant cells to levels comparable to those of control fibroblasts as measured using the pH-sensing ratiometric probe Lysosensor Yellow/Blue DND160. Treatment with non-acidic control NPs did not significantly affect the lysosomal pH in the \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts (Fig. S4B, C). TMRM staining demonstrated that both rapamycin and the acidic NPs significantly rescued the ΔΨm in the mutant fibroblasts (Fig S4F, G). We also found that the ΔΨm was increased in control 2 and E326K fibroblasts upon control NP treatment (Fig S4G). We wondered whether the effect of control NPs on ΔΨm could be due to the succinate component in the poly(ethylene succinate) control NPs. However, treatment with 3mM Diethyl Succinate for 30 minutes did not have any effect on the ΔΨm in control and \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts (Fig S4H).\u003c/p\u003e\u003cp\u003eWe then assessed the impact of rapamycin and acidic NPs on \u003cem\u003eGBA1\u003c/em\u003e mutant iPSC-derived DA neurons (Fig.\u0026nbsp;5). Treatment with 200nM rapamycin and 180 \u0026micro;g/mL acidic NPs restored lysosomal pH, ΔΨm, and mitophagy in the E326K and N370S iPSC DA neurons (Fig.\u0026nbsp;5A, B, C). Although there was no change in GBA1 expression levels, GCase activity was increased in \u003cem\u003eGBA1\u003c/em\u003e mutant DA neurons following treatment with either rapamycin or acidic NPs (Fig S4 D, E). Additionally, western blotting of OXPHOS complexes indicated that the reduced mitochondrial Complex IV levels in E326K DA neurons and the reduced Complex I levels in the N370S DA neurons were normalised upon treatment with either rapamycin or acidic NPs. (Fig S4I).\u003c/p\u003e\u003cp\u003eAs rapamycin restores the assembly of the V-ATPase complex in \u003cem\u003eGBA1\u003c/em\u003e mutant cells (Fig.\u0026nbsp;4D-F), we asked whether the acidic NPs also function through the V-ATPase complex formation. FLIM-FRET experiments conducted on control and acidic NP-treated \u003cem\u003eGBA1\u003c/em\u003e mutant fibroblasts transduced with ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeongreen and ATP6V\u003csub\u003e0\u003c/sub\u003ea3-mScarlet plasmids demonstrated a further increase in the t\u003csub\u003em\u003c/sub\u003e of ATP6V1B2 in both control and mutant fibroblasts treated with acidic NPs, confirming that the acidic NPs operate independently of V-ATPase (Fig S4J).\u003c/p\u003e\u003cp\u003eThus, the data presented here suggest that constitutive phosphorylation of MTORC1 in GBA1-PD cells leads to impaired lysosomal pH and compromised lysosomal proteolytic function. This results in the accumulation of mitolysosomes and impaired mitophagy. The compromised mitophagy culminates in mitochondrial dysfunction in \u003cem\u003eGBA1\u003c/em\u003e mutant cells. Furthermore, inhibiting MTORC1 activity or pharmacologically increasing lysosomal pH using acidic NPs enhances lysosomal proteolytic activity, rescuing mitochondrial dysfunction in \u003cem\u003eGBA1\u003c/em\u003e mutant iPSC-DA neurons and fibroblasts.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe report a range of abnormalities in the lysosomes and mitochondria of GBA1-PD fibroblasts and iPSC-DA neurons carrying E326K or N370S \u003cem\u003eGBA1\u003c/em\u003e mutations. Given that GBA1 is a lysosomal resident enzyme, we hypothesised that mitochondrial dysfunction is likely to reflect a primary lysosomal defect. We used the ratiometric probe Lysosensor Yellow/Blue DND 160 to measure lysosomal pH. When calibrated, these measurements revealed that lysosomal pH in control cells ranged from 4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 to 4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, while in mutant cells it showed a significant shift to a range of 4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 to 5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03. Reports in the literature indicate that the optimal pH for lysosomal function ranges between 4.2 and 4.8, depending on the cell type [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although the difference between control and mutant cells seems modest, the data we present suggest that this difference in mutant cells is functionally significant. In line with other studies, we observed a pH-dependent decrease in the lysosomal enzyme activity, an increase in lysosomal number, and disruption of mitophagy and mitochondrial metabolism [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37 CR38\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVery few reports discuss mitochondrial abnormalities associated with GBA1-PD [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and even fewer discuss mitophagy [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We noted reduced ΔΨm and mitochondrial fragmentation alongside swollen mitochondrial structures, decreased cristae density, and a reduced oxygen consumption rate, all indicating dysfunctional mitochondrial oxidative phosphorylation. This was also associated with reduced mitophagy in the E326K and N370S GBA1-PD fibroblasts and iPSC-DA neurons.\u003c/p\u003e\u003cp\u003eNumerous studies have suggested mechanistic evidence for a connection between PD and \u003cem\u003eGBA1\u003c/em\u003e mutations. For example, dysfunctional mitochondria in primary neurons and astrocytes were described in the \u003cem\u003eGba1\u003c/em\u003e mouse knockout model, associated with an impaired autophagy-lysosomal pathway, resulting in the accumulation of ubiquitinated proteins and α-synuclein[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Various studies have explored the mechanisms behind the PD pathology in \u003cem\u003eGBA1\u003c/em\u003e mutant models. A recent study emphasised that decreased GCase activity in GBA1-PD midbrain dopaminergic neuronal cells leads to defective modulation of the untethering protein TBC1D15, which regulates Rab7 GTP hydrolysis for contact untethering. This impairment results in prolonged mitochondria-lysosome contacts, potentially affecting mitochondrial dynamics and function in GBA1-linked PD[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Baden et al. proposed an alternative function of GBA1 in the mitochondria in maintaining the integrity of complex I and support of energy metabolism[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], although in the present study we found no evidence for mitochondrial localisation of GBA1 nor impaired assembly of complex I.\u003c/p\u003e\u003cp\u003eOur respirometry assays using isolated mitochondria revealed a marked reduction in both maximal respiratory capacity and ATPase-linked oxygen consumption in GBA1-E326K and N370S mutant DA neurons, upon stimulation with complex I (pyruvate/malate) and complex II (rotenone/succinate) substrates. The impairment across both complexes points to a global mitochondrial dysfunction, potentially arising from structural abnormalities such as mitochondrial enlargement and decreased cristae density\u0026mdash;features observed in these mutants\u0026mdash;or as a downstream consequence of lysosomal dysfunction. Notably, lysosome-related mitochondrial impairment has also been reported in Tre-2/Bub2/Cdc16 (TBC1) domain containing Kinase (TBCK) encephaloneuronopathy, supporting a broader link between lysosomal defects and mitochondrial pathology[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent evidence suggests that the primary cause of the pathologies associated with lysosomes and mitochondria in PD is the \u003cem\u003eGBA1\u003c/em\u003e mutation. There appears to be a correlation between the onset of PD and lysosomal dysfunction caused by impaired GCase activity. Dysfunctional lysosomes disrupt cellular signalling networks, metabolic processes, autophagy, and the regulation of calcium (Ca\u0026sup2;⁺) signalling, all of which may contribute to PD pathology[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, both lysosomal function and autophagy are intrinsically dependent on lysosomal acidification, which is influenced by the pH-dependence of lysosomal enzyme activity and substrate degradation, as well as the regulation of lysosomal calcium efflux[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Li et al. also suggest that autophagy in L444P mutant GBA1-PD neurons is impaired at two stages: the initial autophagy stage and the lysosomal degradation stage. While these reports indicate a relationship between \u003cem\u003eGBA1\u003c/em\u003e mutations and autophagic, lysosomal, and mitochondrial dysfunction, a mechanistic dissection of these multi-organellar dysfunctions is lacking.\u003c/p\u003e\u003cp\u003eDysregulation of the MTORC1 pathway is reported in various neurodegenerative diseases, including PD and Alzheimer\u0026rsquo;s disease[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A recent study by Chen et al. suggests that hyperphosphorylation of MTORC1 in striatal inhibitory neurons diminishes the disruption of dopamine receptor activity, alongside odour preference, in transgenic mice [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Liu et al. demonstrate the role of MTORC1 in dopamine dynamics and synaptic plasticity[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These results elucidate the additional impacts of increased phosphorylated MTORC1 on PD. Kaempferol, an MTORC1 inhibitor, has also been shown to promote autophagy and to protect DA neurons in the MPTP-induced C57BL/6J\u0026ndash;PD mouse model [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. All this literature suggests that hyperphosphorylated MTORC1 may play a central role in the development of PD associated with \u003cem\u003eGBA1\u003c/em\u003e mutations. Our study also indicates increased MTORC1 phosphorylation in GBA1-PD fibroblasts and iPSC-DA neurons. While the link between MTORC1 hyperphosphorylation and \u003cem\u003eGBA1\u003c/em\u003e mutations is unclear, it may reflect a cellular stress response to lipid substrate accumulation, indicating nutrient deprivation under \u003cem\u003eGBA1\u003c/em\u003e mutant conditions[\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent evidence underscores the significance of impaired lysosomal acidification in various disease processes. Notably, impaired lysosomal acidification is proposed to be a critical factor in the pathogenesis of multiple neurodegenerative diseases[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Mutations in presenilin-1 (\u003cem\u003ePS1\u003c/em\u003e) associated with familial Alzheimer\u0026rsquo;s disease have been demonstrated to inhibit the lysosomal acidification of human \u003cem\u003ePS1\u003c/em\u003e mutant fibroblasts, resulting in chronic alterations in autophagy and degradation, which may be restored through lysosomal reacidification[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Furthermore, it has been established that individuals with Down Syndrome, who are predisposed to early-onset dementia resembling Alzheimer\u0026rsquo;s disease, exhibit increased levels of the β-cleaved carboxy-terminal fragment of the amyloid precursor protein due to the presence of an additional chromosome 21, which impairs lysosomal acidification and functionality via the inhibition of V-ATPase[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Collectively, these studies illuminate the connections between the impairment of lysosomal acidification in autophagic pathways and the pathogenesis of neurodegeneration, thereby presenting potential therapeutic avenues for targeting lysosomal function and pH to mitigate these debilitating diseases. Acidic NPs in the form of poly(lactic-co-glycolic acid) (PLGA) acidic NPs have been previously used to reduce lysosomal pH and disease progression in cells derived from GBA1-N370S PD patient fibroblasts and in PD mouse models[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Additionally, poly-succinate acidic NPs used in this study have been employed to restore lysosomal pH and rehabilitate metabolic and lysosomal functions in cases of non-alcoholic fatty liver disease[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs an MTORC1 inhibitor[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], rapamycin also acts as a prominent inducer of mitophagy and has been demonstrated to mitigate mitochondrial dysfunction in various disease models through inhibition of the MTOR pathway[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Consistent with findings from other studies, we found that rapamycin rescues mitophagy and mitochondrial function in GBA1-PD fibroblasts and DA neurons as well. This could either be due to the role of MTOR as a regulator of mitochondrial function[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] or a downstream effect of lysosomal acidification by MTOR inhibition. To establish whether impaired lysosomal acidification is the cause of these dysfunctions, we employed an independent pH modulator in the form of novel acidic poly-succinate NPs, which rectify lysosomal pH in the cells while enhancing lysosomal and mitochondrial functions in GBA1-PD fibroblasts and DA neurons. Our data confirmed the efficacy of acidic NP treatment on lysosomal pH \u003cem\u003ein vitro\u003c/em\u003e. We show that the ratio of alkaline vs acidic lysosomes decreased significantly in all mutants and most controls after the acidic NP treatment, demonstrating a decrease in mean lysosomal pH. The effect on controls could be due to the alkalinisation of the lysosomes with age, which can be reversed by the acidic NP treatment in healthy cells[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Importantly, treatment with acidic NP successfully eliminated the differences in lysosomal pH between the \u003cem\u003eGBA1\u003c/em\u003e mutants and the controls. We conclude that the restoration of lysosomal acidification in the mutant cells improved lysosomal function.\u003c/p\u003e\u003cp\u003eLysosomal acidification also rescued GCase activity in the cells despite mutations in the GBA1 protein. Other studies have also demonstrated the restoration of GCase activity despite \u003cem\u003eGBA1\u003c/em\u003e mutations under acidic pH conditions[\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The N370S mutation results in a structurally rigid protein with reduced intralysosomal stability and reduced GCase activity[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]and the E326K mutation has little effect on the active catalytic site of the protein but affects the dimerisation and quaternary structure formation due to folding defects, likely leading to a mild reduction in GCase activity at higher pH levels[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. GCase activity depends on protonation of amino acids at the active sites and due to interaction of the protein with activators like saposin C[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], which is affected due to the mutation[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Restoring the acidic pH in the lysosomes helps restore functional GCase activity despite the mutations possibly by stabilising protein structure, improving folding and optimising the active-site ionisation[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eTo explore whether altered lysosomal pH is related to mitochondrial dysfunction in GBA1-PD, we evaluated ΔΨm, redox levels, and oxidative phosphorylation (OXPHOS) protein levels following treatment with NPs. Our results reveal a significant rescue of ΔΨm among GBA-PD mutants after acidic NP treatment, indicating improved mitochondrial function. When compared to untreated healthy controls, the difference in ΔΨm was no longer statistically significant in the mutants following treatment with acidic NPs. It is noteworthy that some control samples also exhibited alterations due to acidic NP treatment, resulting in a small but significant increase in ΔΨm. We also found a significant increase in ΔΨm in control 2 and E326K fibroblasts upon control NP treatment. Since the NPs are composed of poly(ethylene-succinate), we suspected this could be a substrate-mediated (succinate) effect in control NP treated conditions. However, 30-minute treatment with 3mM Diethyl Succinate did not alter the ΔΨm in the control or mutant fibroblasts. Rescuing lysosomal pH also improved mitophagy in the GBA1-PD fibroblasts and iPSC-DA neurons.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have explored the relationship between lysosome biology, pH, mitophagy and mitochondria in cells carrying PD-associated mutations of \u003cem\u003eGBA1\u003c/em\u003e. A detailed examination of the pathophysiology of these cells reveals a range of abnormalities in the lysosomes, including reduced GCase activity, an increased lysosomal number, impaired lysosomal acidification, and impaired pH-dependent lysosomal proteolytic activity. Mitochondrial dysfunction is reflected by reduced mitochondrial membrane potential, swollen, fragmented mitochondria, decreased cristae density, and the accumulation of abnormal mitochondria, along with a reduced oxygen consumption rate. We also found a decrease in mitophagy and an accumulation of autolysosomes in the GBA1-PD cells. We show that increased pMTORC1 on the lysosomal membrane, possibly due to accumulation of substrates in the lysosomes, causing nutrient deprivation, hinders the formation of a functional V-ATPase complex in GBA1-PD cells, thereby compromising lysosomal acidification. We found that inhibiting MTORC1 using rapamycin rescues functional V-ATPase formation and lysosomal pH in the GBA1-PD neurons and rescues mitophagy and mitochondrial function. Independent modulation of lysosomal pH using acidic poly-succinate NPs, restored lysosomal pH, rescued mitophagy and restored mitochondrial function in the GBA1-PD fibroblasts and iPSC-DA neurons (Fig.\u0026nbsp;6). Since rapamycin has broad effects, the comparison to NPs is valuable, adding a translational value for future applications. We thus unravel the mechanism behind impaired lysosomal pH in GBA1-PD cells and demonstrate that restoring lysosomal pH is sufficient to rescue both lysosomal and mitochondrial dysfunction, along with mitophagy in GBA1-PD fibroblasts and iPSC-DA neurons.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and ethics committee approval:\u003c/h2\u003e\u003cp\u003eThe human iPSC (hiPSC) and fibroblast lines used in the study, along with their sources, are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Fibroblasts were cultured in DMEM (1x)\u0026thinsp;+\u0026thinsp;GlutaMAX\u0026trade; basal media (Gibco 2206106), 10% (v/v) Foetal Bovine Serum (Gibco 10270098), and 1% antibiotic-antimycotic (Gibco 15240096), and incubated at 37 \u0026ordm;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The media was changed every 72 hours, and the cells were passaged upon reaching 80% confluency using 0.25% Trypsin-EDTA (Gibco 25200056). Passages between 9 and 15 were used for experimental purposes. Human GBA1 patient-derived iPSC lines were cultured under feeder-free conditions on Geltrex-coated 6-well plates, using mTeSR Plus medium and were passaged every 4\u0026ndash;5 days with a 0.5 mM EDTA solution or upon reaching 80% confluency. Passage numbers between 20 and 30 were employed for differentiation purposes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHuman pluripotent stem cell-derived DA neurons\u003c/h2\u003e\u003cp\u003eDA neurons were generated from iPSCs using a step-by-step differentiation protocol, as previously described[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Briefly, 90\u0026ndash;95% confluent hiPSCs were cultured on Geltrex-coated plates and maintained in N2B27 medium, which is a 1:1 mix of Neurobasal (Gibco, 21103049) supplemented with 1X B27 (Gibco, 17504044), Glutamax (Gibco, 35050-038), and 0.5X Anti-anti (Gibco, 15240096), and DMEM-F12 (Gibco, 10565018) supplemented with 1X N2 supplement (Gibco, 17502048), 1X MEM Non-Essential Amino Acids (Gibco, 11140-050), 0.5X Anti-anti (Gibco, 15420096), 50 \u0026micro;M β-mercaptoethanol (Gibco, 21985-023), and human Insulin Solution (Sigma, I9278). Daily media changes were conducted for the first 14 days. On day 0, the media was supplemented with 5 \u0026micro;M SB431542 (Tocris Bioscience, 1614/10), 2 \u0026micro;M Dorsomorphin (Tocris Bioscience, 3093/10), and 1 \u0026micro;M CHIR99021 until day 2. From day 2 to day 7, 1 \u0026micro;M Purmorphamine (Merck Millipore, SML0868) was added to the mix. From day 8 to day 14, CHIR99021 and SB431542 were removed from the medium, and the cells were maintained with Dorsomorphin and Purmorphamine in N2B27. Cells were dissociated with 1 mg/mL Dispase (Gibco, 17105041) on days 4 and 14. After patterning for 14 days, the midbrain DA neuronal precursor cells (NPCs) were maintained in N2B27 medium until day 18. On day 19, the cells were dissociated with Accutase (Gibco, A1110501) and plated onto Geltrex-coated plates at a density of 2 \u0026times; 10^5 cells/cm\u0026sup2; and terminally differentiated with N2B27 medium supplemented with Compound E (Enzo Lifesciences, ALX-270-415-C250) and 10 \u0026micro;M Rho-kinase inhibitor, Y-27632 dihydrochloride (Tocris, 1254) from day 20 until day 70\u0026ndash;75, with media changes twice a week. DA neurons between days 70 and 75 were used for downstream experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNanoparticle, rapamycin and diethyl succinate treatment\u003c/h2\u003e\u003cp\u003eControl and acidic NPs, composed of poly(ethylene-succinate) and poly(ethylene tetrafluorosuccinate-co-succinate) respectively, were obtained from Prof. Mark Grinstaff and Prof. Orian Shirihai. The synthesis of the nanoparticles is described in [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The nanoparticles were stored at -80\u0026deg;C in aliquots and thawed fresh prior to treatment. Aliquots of 75 mg/mL nanoparticles were thawed to room temperature, vortexed thoroughly, and added to the cells at a concentration of 180 \u0026micro;g/mL in fresh culture medium for 12\u0026ndash;16 hours before the experiment. A stock solution of 200 \u0026micro;M rapamycin (Sigma, R0395) was prepared in DMSO and sterile filtered before being stored as aliquots at -20\u0026ordm;C. This stock solution was diluted to 200 nM in medium before being added to the cells. The cells were treated with 200 nM rapamycin for 12\u0026ndash;16 hours before proceeding with the experiments unless mentioned otherwise. Diethyl Succinate (Merck, 112402) was diluted to 3 mM in culture media and sterile-filtered and added onto cells 30 minutes prior to measuring the ΔΨm using TMRM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of lentivirus and transduction:\u003c/h2\u003e\u003cp\u003epLV-Ef1a-3xHA-ATP6V1B2-mNeonGreen and pLV-Ef1a-3xFlag-ATP6Voa3-mScarlet plasmids were kindly provided by Dr. Wilhelm Palm (EPR 462 and EPR 475, European Plasmid Repository). pHAGE-mt-mKeima was a generous gift from Prof. Richard Youle (131626, Addgene). Lentiviral plasmids were packaged using psPAX2 and pMD2.G in HEK293T cells with Xtremegene-HP transfection reagent (Merck, 6366236001), according to the manufacturer\u0026rsquo;s instructions. The media supernatant containing viruses was collected 48 and 72 hours post-transfection and concentrated using Lenti-X transfection reagent (Takara, 631231) with overnight incubation, followed by centrifugation at 2000 rpm for 1 hour at 4\u0026ordm;C. The pellet was then resuspended in DMEM-F12 medium and stored at -80\u0026ordm;C for further use. 72\u0026ndash;96 hours prior to imaging, fibroblasts or DA neurons were treated with lentiviral particles. 24 hours post-treatment, the media was replaced with regular culture medium and incubated for another 48 hours before imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eConfocal imaging\u003c/h2\u003e\u003cp\u003eDMEM-F12 medium (Gibco, 21041025) or BrainPhys medium (Stem Cell Technologies, 05796) served as a recording buffer for live cell imaging of fibroblasts and DA neurons. All images were acquired using a Carl Zeiss LSM 880 Confocal Laser Scanning microscope with Zen Black Software and a 63\u0026times;/1.40 oil immersion lens at 37\u0026deg;C. Fixed and stained fibroblasts were also imaged with the same equipment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLysosensor Yellow/Blue DND 160\u003c/h2\u003e\u003cp\u003eThe cells were washed with DPBS and incubated for 4 minutes in the recording buffer containing 3 \u0026micro;M Lysosensor Yellow/Blue DND 160 (Invitrogen, L7545) at 37\u0026deg;C. After incubation, the dye was washed twice with PBS and imaged in fresh recording buffer using the UV laser at 355 nm. The dual emission maxima of the dye (440 nm/540 nm) were detected using spectral scanning (lambda scan mode) on the Zeiss LSM 880. Images were subsequently linearly unmixed employing the ZEN Black software to separate the 440 nm and 540 nm emission images. The ratio of 440/540 nm was then calculated using the Ratio Plus plugin on ImageJ/Fiji software to determine the lysosomal acidity of fibroblasts and DA neurons\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLysosomal pH measurement\u003c/h2\u003e\u003cp\u003eThe lysosomal pH was measured using Lysosensor Yellow/Blue DND-160 (L7545, Invitrogen). Fibroblasts cultured on fluorodishes were treated with pH calibration buffer containing Monensin (Sigma, M5273) and Nigericin (Sigma, N7143), with a pH ranging from 3.5 to 7.0 and stained with Lysosensor Yellow/Blue DND 160. The cells were incubated with 2 \u0026micro;M for 5 minutes at room temperature (RT) and imaged as previously mentioned. The 440/540 fluorescence ratio was measured for each calibration buffer. A lysosomal pH calibration curve was established by correlating the 440/540 nm ratio with the corresponding pH values using ImageJ/Fiji software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of lysosomal proteolytic activity by DQ Red BSA\u003c/h2\u003e\u003cp\u003eThe DQ-Red BSA trafficking assay dye (Invitrogen, D12051) was utilised to examine lysosomal proteolytic activity. DA neurones were washed with DPBS, and pre-warmed medium containing DQ-Red BSA was added to the fluorodishes. The dishes were incubated at 37\u0026deg;C for three hours. Following the incubation period, the cells were washed with DPBS and replaced with the recording buffer prior to imaging. Cells stained with DQ-Red BSA were excited using a 561 nm Argon laser, and the emitted fluorescence was collected within the 564\u0026ndash;740 nm range. The fluorescence intensity was quantified per cell using ImageJ/Fiji software with consistent threshold settings across all samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of ΔΨm using TMRM\u003c/h2\u003e\u003cp\u003eFibroblast or DA neurons were washed with DPBS and incubated in recording buffer containing 20 nM TMRM (Thermofisher Scientific, T668) at 37\u0026deg;C for 30 minutes. Following incubation, fresh recording buffer containing 20 nM TMRM was added before proceeding with imaging. The cells were excited for TMRM fluorescence at 561nm, and images were acquired as Z-stacks. Maximum intensity projection images were then utilised to quantify the fluorescence intensity using ImageJ/Fiji software with consistent threshold settings across all samples. The TMRM images were also used to analyse the mitochondrial morphology [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of Lipofuscin levels\u003c/h2\u003e\u003cp\u003eLipofuscins are excited at 355 nm and fluoresce across a range from 480 to 700 nm[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Fibroblasts were washed with the recording buffer and imaged using UV illumination at 355 nm, with images obtained through spectral scanning. The images were subsequently linearly unmixed using ZEN Black software to separate the 460 nm and 480 nm emission images, thus distinguishing NAD(P)H from lipofuscins. The spectrally unmixed images at 480 nm were then utilised to calculate lipofuscin density per cell using ImageJ/Fiji software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eColocalization of the Rhodamine-B labelled aNPs with the lysosomes\u003c/h2\u003e\u003cp\u003eRhodamine B-labelled acidic nanoparticles were treated for 12\u0026ndash;16 hours prior to imaging the fibroblasts. On the imaging day, cells were washed with the recording buffer and incubated with 50 nM LysoTracker Blue DND-22 dye (Invitrogen, L7525) for 2 hours at 37\u0026deg;C. Following the incubation, the cells were washed with the recording buffer and imaged in fresh buffer. Images were acquired using sequential excitation (405 nm for LysoTracker Blue and 561 nm for Rhodamine) and emission ranges set to 420\u0026ndash;480 nm and 570\u0026ndash;620 nm, respectively. Colocalisation Pearson\u0026rsquo;s coefficient was quantified using ImageJ/Fiji software with consistent threshold settings across all samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of mitophagy using mt-Keima reporter\u003c/h2\u003e\u003cp\u003eMeasurement of mitophagy was performed as previously described[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Untreated fibroblasts and DA neurons were tranduced with lentiviral mt-Keima particles 72 hrs prior to imaging. 12\u0026ndash;16 hours before imaging, the cells were treated with acidic nanoparticles or rapamycin. The cells were imaged using two sequential excitation wavelengths (458 nm for green fluorescence and 561 nm for red fluorescence) with an emission range of 570\u0026ndash;695 nm. The laser power was set at the minimum output to allow the clear visualization of the mt-Keima signal. The ratio of the high F\u003csub\u003e543\u003c/sub\u003e:F\u003csub\u003e458\u003c/sub\u003e ratio values were generated using the Ratio Plus plugin in ImageJ/Fiji and was used as an index of mitophagy.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eFluorescence lifetime imaging microscopy (FLIM) quantification of ATP6V\u003csub\u003e1\u003c/sub\u003eB2-mNeonGreen\u003c/h2\u003e\u003cp\u003eFluorescence lifetime imaging was conducted using single-photon excitation on a multimodal time-resolved fluorescence microscope as previously described[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. This setup encompassed an 80 MHz, near-infrared, femtosecond excitation source (Insight X3, Spectra Physics, Crewe, UK), a second harmonic generation unit (Harmonixx SHG, APE, Berlin, Germany), a laser scanning unit (DCS-120, Becker \u0026amp; Hickl, Berlin, Germany), an inverted microscope (Axio Observer 7, Zeiss, Cambridge, UK) featuring a high numerical aperture objective (Plan-Apochromat 63x/1.4 Oil M27, Zeiss, Cambridge, UK), an ultrafast hybrid detector (HPM-100-07, Becker \u0026amp; Hickl, Berlin, Germany), and time-correlated single photon counting (TCSPC) electronics (SPC-180NX, Becker \u0026amp; Hickl, Berlin, Germany). Images were acquired using 473 nm excitation to minimise the ratio of acceptor to donor excitation, along with 500\u0026ndash;540 nm emission filtering to isolate fluorescence from the mNeonGreen donor. Photon counts were acquired for two minutes and histogrammed at 14.6ps intervals. Curve fitting analysis was performed in SPCImage (Becker \u0026amp; Hickl, Berlin, Germany).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLysosomal enrichment assay\u003c/h2\u003e\u003cp\u003eA lysosomal enrichment assay was conducted using the Lysosome Enrichment Kit for Tissues and Cultured Cells (Thermo Scientific, 89839) according to the manufacturer\u0026rsquo;s instructions. Briefly, the DA neurons were pelleted and lysed with lysosome enrichment reagent A, which contained 1X protease and phosphatase inhibitors. The solution was then sonicated on ice, applying 12 bursts at 9W of power (Thermo Scientific) and mixed with lysosome enrichment reagent B. The solution was subsequently centrifuged at 500g for 10 minutes. 200 \u0026micro;L of supernatant was then aliquoted for use as cell supernatant for western blotting. Lysosomes were isolated from the remaining solution through gradient centrifugation. The protein concentrations of the lysosomes and cell supernatant were then quantified using a BCA assay and proceeded for western blotting as discussed below.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eSDS-PAGE and immunoblotting\u003c/h2\u003e\u003cp\u003eFibroblasts and DA neurons were washed with ice-cold PBS, followed by the addition of 150 \u0026micro;l of ice-cold RIPA lysis buffer (Sigma-Aldrich, R0278), which was supplemented with protease inhibitors (Roche 4693116001), PMSF (Sigma, 93482), and phosphatase inhibitors (Roche 4906837001). Cells were scraped using a plastic scraper, and the lysates were transferred to 1.5 ml tubes. The lysates were rotated at 4\u0026deg;C for 30 minutes and sonicated (3 cycles, 3 seconds each at 40% amplitude, with 5-minute intervals). Samples were centrifuged at 16,000 g for 30 minutes at 4\u0026deg;C, and the supernatant was collected. Protein concentration was determined using a BCA assay kit (Thermo Scientific, 23227). A total of 20\u0026ndash;30 \u0026micro;g of protein was diluted with RIPA buffer and mixed with NuPAGE 4X sample buffer (Invitrogen, NP0007). The samples were heated at 95\u0026deg;C for 5 minutes (for OXPHOS proteins, the lysates were heated at 45\u0026ordm;C for 5 minutes). Proteins were separated on 4\u0026ndash;12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen, NP0335) immersed in MOPS running buffer (Invitrogen, NP0001). Proteins were transferred to PVDF membranes (Millipore, IPFL00010) activated in methanol using a wet transfer system. Membranes were blocked in Superblock blocking buffer (Invitrogen, 37545) for 1 hour at room temperature. The blots were cut where necessary before incubating with primary antibodies, which were diluted in 1X blocking buffer, and incubated with the membranes overnight at 4\u0026deg;C. After three 10-minute washes in TBST, the membranes were incubated with secondary antibodies (Li-COR Biosciences; 1:10000; IRDye\u0026reg; 680RD Goat anti-Mouse IgG, 926-68070; IRDye\u0026reg; 800CW Goat anti-Rabbit IgG, 926-32211) diluted in 1% BSA/TBST for 1 hour at room temperature. The membranes were washed three additional times with TBST. Fluorescent signals were developed using a LiCOR Odyssey CLx system. The details of the antibodies are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of antibodies used in the study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCatalogue no.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHost\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCompany\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eApplication\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATP6V0D2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAB194557\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLAMP1 (H4A3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esc-20011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSanta Cruz\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting, Immunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGBA (c-term)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSigma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:700\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATP6V1H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAB187706\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATP6V1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e199326\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epMTOR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55365\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMTOR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCell Signalling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATG5 (D5F5U)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12994T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCell Signalling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e610833\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBD Biosystems\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL7543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSigma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-ACTIN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eab8226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCell Signalling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:5000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTOM20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eab186735\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePM036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMBL Biosystems\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitrate Synthetase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eab96600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRabbit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:200\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOXPHOS Cocktail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45-8199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThermoscientific\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlexa Fluor 488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA-11008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGoat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThermoscientific\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlexa Fluor 594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA-11012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGoat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThermoscientific\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlexa Fluor 647\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA-21235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGoat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThermoscientific\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImmunofluorescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIRDye\u0026reg; 680RD Goat anti-Mouse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e926-68070\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGoat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLi-COR Biosciences\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:10000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIRDye\u0026reg; 800CW Goat anti-Rabbit IgG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e926-32211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGoat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLi-COR Biosciences\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWestern Blotting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1:10000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eImmunostaining\u003c/h2\u003e\u003cp\u003eFibroblasts were grown on coverslips, treated, and fixed with 4% (w/v) paraformaldehyde. After fixation, the cells were permeabilised with 50 \u0026micro;g/ml digitonin in PBS for 10 minutes. The cells were then washed, blocked with 3% BSA, and incubated with the following primary antibodies: citrate synthase, LAMP1, and LC3 in 3% BSA for 1 hour at room temperature, followed by incubation with Alexa Fluor 488/594/647-conjugated secondary antibodies for 1 hour at room temperature. Coverslips were mounted on glass slides, and images were acquired using appropriate excitation and emission filters to capture fluorescent signals. Colocalisation Pearson\u0026rsquo;s coefficient (Mito vs LC3 and Mito vs LAMP1), lysosomal number (LAMP1 particles/cell), and autophagosome number (LC3 particles/cell) were quantified using ImageJ/Fiji with consistent threshold settings across all samples. The catalogue number and dilution range of the antibodies are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMitochondrial isolation\u003c/h2\u003e\u003cp\u003eMitochondria were isolated according to the method described earlier [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] and modified for the neuronal cultures. Briefly, DA neuron cell pellets were resuspended in mitochondria isolation buffer (MIB1: 225 mM Mannitol, 75 mM Sucrose, 5 mM HEPES, 1 mM EGTA and 1 mg/ml fatty acid free BSA) and homogenised using a ice-cold Dounce tissue grinder tube (appropriate for 1-1.5 ml homogenisation volume). The homogenate was centrifuged, and the mitochondrial pellet was washed and pellet down in MIB without BSA (MIB2) according to the protocol. The final pellet was resuspended in a very small volume of MIB2, and the protein concentration was determined using BCA Protein assay kit (Thermofisher Scientific, 23227) according to the manufacturer\u0026rsquo;s specifications.The isolated mitochondria were then used for respiratory measurements.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of oxygen consumption rate\u003c/h2\u003e\u003cp\u003eMeasurements of mitochondrial respiration were conducted with the Seahorse Bioscience XFe96 bioanalyzer using the Seahorse XF Cell Mito Stress Test Kit (Agilent #103015-100). Final maturation of iPSCs into DA neurons were performed in XF96 cell culture microplates (Agilent #102416-100). On the day of the experiment, the culture medium was replaced with Seahorse XF Base medium (Agilent #103334-100) supplemented with 1 mM pyruvate (Gibco #11360070), 2 mM glutamine (Gibco #25030081) and 10 mM glucose (Gibco #A2494001) and incubated for 30 min at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e-free incubator before loading into the Seahorse Analyser. After measuring basal respiration, the drugs oligomycin (5 \u0026micro;M), FCCP (1.5 \u0026micro;M), and rotenone/antimycin A (0.5 \u0026micro;M/0.5 \u0026micro;M) were added to each well in sequential order. After the assay, cells were stained with Hoechst 33342 (5 \u0026micro;M; Thermo Scientific #62249) for 30 min. ImageXpress was then used to count the number of cell nuclei (cell numbers) in each well and normalised to get the basal respiration rate values.\u003c/p\u003e\u003cp\u003eThe respiratory measurements of isolated mitochondria from mixed neuronal cultures were performed using a modified method described earlier[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] for Seahorse XFe96 cell culture plates assay. Briefly, 5 or 10 \u0026micro;g of mitochondrial protein was resuspended in 30 \u0026micro;L (1 well) of individual substrate mix (for example, pyruvate/malate substrate\u0026thinsp;+\u0026thinsp;mitochondria assay solution - MAS) and plated into each well. The cell culture plate was centrifuged at 2,000g for 20 minutes at 4\u0026deg;C to form a uniform layer of mitochondria at the bottom. After centrifugation, 150 \u0026micro;L of substrate solution was carefully added to each well. Fresh injection solutions were made in MAS without BSA and loaded into the cartridge, and calibrated according to the manufacturer\u0026rsquo;s specifications. After calibration, the culture plate with mitochondria was inserted, and the assay was run essentially as described in the method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e\u003cp\u003eTEM was performed as previously described [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Briefly, fibroblasts and DA neurons grown on coverslips were fixed in electron microscopy (EM) fixative containing 2% glutaraldehyde (EMS, 16365) and 2% paraformaldehyde (EMS, 15710) in 0.1 M sodium cacodylate for 1 hour. Following fixation, cells were washed with 0.1 M cacodylate buffer (EMS, 11650) and then fixed in a solution of 1% osmium tetroxide (EMS, 19150) and 1% potassium ferricyanide (EMS, 25120-20) in 0.1 M sodium cacodylate. This was followed by sequential dehydration using ethanol. Coverslips were then embedded in epoxy resin (Araldite Kit, Agar Scientific Ltd., CY212) according to standard protocols. The embedded samples were sectioned into 50 nm slices using an ultramicrotome equipped with a diamond knife and mounted onto copper grids suitable for TEM. The grids were stained with lead citrate for 3 minutes before imaging. Images were captured using a Jeol 1400 Transmission Electron Microscope at magnifications ranging from 800\u0026times; to 1200\u0026times; (digital magnification). Images were imported into ImageJ/Fiji to quantify mitochondrial area, mitochondrial cristae density, and the number of autophagosomes, autolysosomes, and lysosomes per field. Mitochondria and autophagic vesicles were manually traced to determine the area.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantification and statistical analysis\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism. For comparisons between two groups, a two-tailed unpaired t-test was applied to normally distributed data. Multi-group comparisons were analysed using one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test or two-way ANOVA. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. from at least three independent replicates, with 50\u0026ndash;70 cells per repeat for DA neurons and 10\u0026ndash;15 cells per repeat for fibroblasts for live cell imaging experiments. For immunoblotting or TMRM data, control sample means were normalised to one to facilitate comparisons. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. p-values are reported as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns \u0026ndash; not significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGBA1\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlucocerebrocidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParkinson\u0026rsquo;s Disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGCase\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eβ-glucocerebrosidase enzyme\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003epH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epotential of Hydrogen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eV\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cb\u003eATPase\u003c/b\u003e-Vascular ATPase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eiPSCs\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einduced Pluripotent Stem Cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDA neuron\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDopaminergic neurons\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eOXPHOS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOxidative Phosphorylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eΔΨm\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitochondrial Membrane Potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTMRM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTetraMethyl-Rhodamine Methyl ester\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eMTORC1\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMammalian Target of Rapamycin Complex I\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFLIM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cb\u003eFRET\u003c/b\u003e-Fluorescence Lifetime Imaging (FLIM) with F\u0026ouml;rster Resonance Energy Transfer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNPs\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNanoparticles\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cp\u003eThe source and ethical approval committee for the iPSCs used in the study is tabulated in table 1.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003e\u003cem\u003eNot applicable\u003c/em\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declared no competing interests in this research.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis project was funded by the Michael J Fox Foundation (Project number E27234 to MRD) and the Parkinson\u0026rsquo;s UK Foundation (G-2103 to MRD and PS).\u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e\u003cp\u003eP.S., M.R.D - Conceptualisation, funding acquisition, investigation, visualization, methodology, writing\u0026ndash;original draft, project administration, writing\u0026ndash;review and editing. A.C.B \u0026ndash;methodology, investigation, visualisation and data analysis. S.K, A.F, I.K, K.S and T.S.B\u0026ndash; Methodology, investigation and data analysis. O.S, J.Z and M.G \u0026ndash; Resources.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors would also like to express their gratitude to the members of the Michael Duchen and Gyorgy Szabadkai labs for their feedback, as well as to students, including Shail Bhatt and Oriane Marguet, for their assistance with the preliminary data. We acknowledge the MRC Centre for Neuromuscular Diseases Biobank (supported by the National Institute for Health Research Biomedical Research Centres at Great Ormond Street Hospital for Children, NHS Foundation Trust) for providing the age- and sex-matched healthy controls and GBA1-E326K fibroblasts used in this study. Figure\u0026nbsp;6 was created using Biorender.com. We thank Dr. Elizabeth Slavik-Smith from the Electron Microscopy facility at UCL, Division of Biosciences, for her assistance with EM.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSidransky E, Nalls MA, Aasly JO, Aharon-Peretz J, Annesi G, Barbosa ER, et al. Multicenter Analysis of Glucocerebrosidase Mutations in Parkinson\u0026rsquo;s Disease. N Engl J Med. 2009;361:1651\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFurderer ML, Hertz E, Lopez GJ, Sidransky E. Neuropathological Features of Gaucher Disease and Gaucher Disease with Parkinsonism. IJMS. 2022;23:5842.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLwin A. Glucocerebrosidase mutations in subjects with parkinsonism. 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J Vis Exp. 2015;e53216.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"GBA1, Parkinsons Disease, mitochondria, lysosomes, lysosomal pH, MTOR, acidic nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-7558589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7558589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHeterozygous mutations in the Glucocerebrosidase gene (\u003cem\u003eGBA1\u003c/em\u003e), which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are a genetic risk factor for Parkinson\u0026rsquo;s disease (PD), characterised by lysosomal dysfunction. The pathological effects of \u003cem\u003eGBA1\u003c/em\u003e mutations on PD, especially their influence on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eFibroblasts and dopaminergic neurons, generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD, were used in the study. Live-cell imaging was performed to assess lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial cristae density and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was evaluated using FLIM-FRET, and pharmacological interventions included rapamycin and acidic nanoparticles. Statistical analyses involved unpaired t-tests, one-way ANOVA, and two-way ANOVA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. MTORC1 was constitutively phosphorylated and FLIM-FRET measurements confirmed impaired V-ATPase assembly, which was reversed following rapamycin treatment. Rapamycin and lysosome-specific acidic nanoparticles rescued lysosomal pH, restored mitophagy and mitochondrial membrane potential in \u003cem\u003eGBA1\u003c/em\u003e mutant dopaminergic neurons.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings identify lysosomal acidification as the primary cause of impaired bioenergetic function and reduced mitophagy in GBA1-PD. MTORC1-mediated disruption of V-ATPase assembly drives these pathogenic processes. Pharmacological interventions that restore lysosomal pH\u0026mdash;such as rapamycin or acidic nanoparticles\u0026mdash;rescue both lysosomal and mitochondrial defects, offering a promising therapeutic approach for GBA1-PD.\u003c/p\u003e","manuscriptTitle":"Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 06:46:36","doi":"10.21203/rs.3.rs-7558589/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-23T06:38:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-23T02:23:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T04:51:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Neurodegeneration","date":"2025-09-10T07:30:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tneu","sideBox":"Learn more about [Translational Neurodegeneration](http://translationalneurodegeneration.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/tneu/default.aspx","title":"Translational Neurodegeneration","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c9f1db7-aed2-463b-9717-e5820f249546","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T01:00:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 06:46:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7558589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7558589","identity":"rs-7558589","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}