Long-term oral regimen of glucocerebrosidase activator reduces a-synuclein oligomer accumulation in aged LRRK2 mutant mouse brains - therapeutic implication of Parkinson’s disease

Long-term oral regimen of glucocerebrosidase activator reduces a-synuclein oligomer accumulation in aged LRRK2 mutant mouse brains - therapeutic implication of 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 Article Long-term oral regimen of glucocerebrosidase activator reduces a-synuclein oligomer accumulation in aged LRRK2 mutant mouse brains - therapeutic implication of Parkinson’s disease Zoe Yuen-Kiu Choi, Huifang Liu, Eunice Eun-Seo Chang, Shirley Yin-Yu Pang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7185990/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in npj Parkinson's Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Deposition of misfolded a-synuclein (a-syn) aggregates in brain is a pathological hallmark of Parkinson’s disease (PD). Accumulation of toxic soluble a-syn seeds in patient cerebrospinal fluid represents a prodromal marker of synucleinopathies in PD, contributing to progressive neurodegeneration. Deficiency in beta-glucocerebrosidase (GCase), a lysosomal enzyme for glucocerebroside metabolism, is evident in PD linking functionally with pathogenic LRRK2 (leucine-rich repeat kinase 2) mutation and synucleinopathies. However, whether GCase activation ameliorates synucleinopathies in PD brains is unclear. Here, we explored how GCase activity affected Ser129-a-syn phosphorylation, and whether long-term treatment of a brain-penetrant GCase chaperone (Ambroxol; ABX) attenuated a-syn oligomer accumulation in aged mutant LRRK2 R1441G mouse brains. Acute ABX treatment (50µM) significantly increased cellular GCase activity and reduced Ser129-a-syn phosphorylation in human SH-SY5Y neuronal cells and murine fibroblasts. Real-time DQ-BSA degradation assay revealed lysosomal dysfunction in mutant LRRK2 R1441G MEFs, which was attenuated by ABX treatment. Single oral gavage of ABX (400mg/kg) in mice achieved peak drug level in serum and brain within 6 hours post-administration. Spontaneous feeding of ABX in food pellet over 18 weeks (average daily dose: 45.9mg/kg/day) elevated brain GCase activity in aged wildtype and mutant striatum without affecting body weight. This chronic regimen significantly reduced a-syn oligomer level in mutant striatum yet without an effect on total a-syn and Ser129-phosphorylation levels. This is the first study demonstrating attenuation of synucleinopathies by chronic GCase activation in aged mouse brains vulnerable to PD, suggesting early intervention to alter progression of synucleinopathies as a key determinant of clinical outcomes of PD. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease LRRK2 mutation alpha-synuclein synucleinopathies beta-glucocerebrosidase oligomers Ambroxol GCase activator Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons, associated with characteristic motor and non-motor symptoms. The pathological deposition of misfolded alpha-synuclein (α-syn) protein in the disease brain involves prior post-translational modification (e.g., phosphorylation) and progressive aggregation into toxic higher-molecular-weight soluble oligomers and insoluble fibrils (in form of Lewy bodies). These events collectively contribute to neuronal toxicities that hasten disease progression as the central hallmark of PD 1 . Despite that a number of genetic mutations have been definitively linked to the onset of the disease 2 , the common mechanism involved remains poorly understood. Current treatment options offer only symptomatic relief without halting neurodegeneration. Disease-modifying strategies to delay progression remains the most plausible approach. Recent research has identified new genetic risk factors associated with PD. The most prominent one is GBA1 (GBA; NCBI Gene ID: 2629) mutations 3 . Approximately 5-10% of PD patients among different populations worldwide were found to carry GBA mutations, making this gene one of the most important genetic predisposing risk factors. Glucocerebrosidase (GCase) is a lysosomal enzyme encoded in GBA1 gene which is responsible for the breakdown of glucocerebroside (GlcCer) to glucose and ceramide. GCase deficiency can lead to the accumulation of toxic glycosphingolipids which potentiate α-syn aggregation with yet-unclear mechanism(s) 4,5 . Experimental augmentation of GCase activity in mouse neurons has been shown to modulate pathological α-syn insults 6 . Whilst this evidence supported an association between GCase dysfunction and α-syn aggregation, the beneficial effects of modulating GCase activity in PD patients await further validation. As a native presynaptic protein, α-syn exists in a dynamic equilibrium amongst different conformations 7 . Growing evidence supported that the soluble oligomeric species of α-syn contribute to synucleinopathies in PD 8 . Various detection techniques of synucleinopathies were recently developed and validated under clinical settings. For instances, α-synuclein seed amplification assays (SAA), including real-time quaking-induced conversion (RT-QuIC), can now detect synucleinopathies from newly-diagnosed PD patient cerebrospinal fluid (CSF) samples with high sensitivity and specificity 9 . Moreover, new α-syn PET tracers were introduced to visualize α-syn pathologies in prodromal PD brains 10,11,12 . These diagnostic tools revealed persuasive clinical evidence that clarified the paradox of soluble seed-competent α-syn oligomers in PD pathogenesis and progression 13 . Leucine-rich repeat kinase-2 (LRRK2) mutations represent one of the most common genetic risks in both familial (5-13%) and sporadic (1-5%) PD. Clinically, LRRK2 mutation carriers who develop PD are largely indistinguishable from late-onset idiopathic cases 14 , implying that LRRK2-PD share common disease mechanisms. This protein encodes for two distinctive enzymes, a protein kinase and a putative GTPase, within a single polypeptide chain, serving as an important intracellular signal transduction protein. The LRRK2 R1441G (4321C>G) mutation causes hyperactive kinase activity, and it adversely affects GCase activity that compromised lysosomal function and neuronal survival 15,16 . Two recent independent studies revealed increased α-syn in postmortem brain tissue from LRRK2-associated PD, resembling oligomeric accumulations rather than typical fibrillar Lewy body inclusions 17,18 . Selective loss of GCase activity is associated with the aggregation of α-syn and increased accumulation of α-syn oligomers in sporadic PD patients 19 . Given this, we hypothesize that long-term activation of GCase in the brain may be a viable approach to attenuate the accumulation of α-syn oligomers as induced by pathogenic LRRK2 mutation in PD. Ambroxol (ABX) (trans-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol; CAS number 18683-91-5) is a common medication for respiratory conditions, being utilized as a potential pharmacological chaperone for GCase 20 . Its therapeutic role in inducing GCase activity has been widely investigated, particularly in the context of Gaucher Disease (GD) and PD 20 . Oral administration of ABX is effective to penetrate through the blood-brain barrier (BBB) in nonhuman primates, resulted in increased GCase activity in key brain regions including cortex and striatum 21 . In our earlier studies, we revealed α-syn oligomer accumulation in cortex and striatum of LRRK2 R1441G mutant mice starting at 14 months of age (equivalent to 38–47 years old in human) and significantly increased by 18 months (equivalent to 56–69 years old in human), compared to their age-matched WT littermates. We found that long-term therapeutic inhibition of mutant LRRK2 kinase hyperactivity in these mutant mice over 18 weeks significantly reduced brain α-syn oligomer level 22 . Here, we aimed to use this model to test our hypothesis whether long-term spontaneous feeding of GCase activator (ABX) could attenuate accumulation of toxic α-syn oligomers in aged LRRK2 R1441G mutant mouse brain by inducing brain GCase activity and lysosomal degradation. Our findings further validated the importance of α-syn oligomers as a promising modifiable therapeutic target in the development of PD therapies. Results Treatment of GCase activator (ABX) increased cellular GCase activity and reduced level of Ser129-α-syn phosphorylation without affecting total a Syn expression Reduced GCase activity in PD is linked to α-syn oligomerization and aggregation 19 . In particular, phosphorylation of α-syn at residue Ser129 (pSer129) has been proposed to be a surrogate marker of synucleinopathies in PD 23 . We initially determined whether inducing cellular GCase activity by therapeutic GCase activator, ABX, could modulate Ser129-α-syn phosphorylation in human neuronal cells. Normal SH-SY5Y cells express α-syn protein at very low level. To better elucidate the ABX effect, cells were engineered to stably overexpress WT α-syn by lentivirus transduction and selection. For cell treatment, cells at 70% confluence were refreshed with new medium for 2 hr before ABX treatment at 0, 10 and 50 µM for an additional 72 hr ( Fig. 1a ). Total cellular GBA levels and GCase activity were determined by Western blots and standard 4-MU fluorescence assay in the total cell lysates, respectively. Dose-dependent increase of GCase activity and GBA protein expression was observed in both normal and α-syn overexpressing cells (all p<0.05; N=7), ranging from 10 to 50µM ABX treatment (all p<0.05; N=7) ( Fig. 1b, c ). The cellular GCase activity in 50µM ABX treatment on both normal and α-syn overexpressing cells were significantly increased by 24.2% and 16.9% compared to DMSO-treated controls (p<0.01; N=7) ( Fig. 1b ). Moreover, GBA protein levels in normal and α-syn overexpressing cells were determined by western blot, showing significantly increased by 51.6% (p<0.01) and 33.4% (p<0.05) compared to DMSO-treated controls, respectively ( Fig. 1c ). Furthermore, western blot analysis showed that pSer129-α-syn levels in cells treated with ABX at 50 µM were significantly reduced by 16.9% compared to DMSO-treated controls (p<0.01; N=7) ( Fig. 1c ). Total α-syn level was not affected by ABX treatment at all doses indicating that the reduction of pSer129-α-syn levels were not due to changes in total cellular protein levels ( Fig. 1c ). ABX rescued GCase deficiency and suppressed Ser129-α-syn phosphorylation in LRRK2 R1441G mutant mouse embryonic fibroblasts (MEFs) The property of ABX to reduce Ser129-α-syn phosphorylation in human SH-SY5Y cells (Fig. 1) highlighted the therapeutic potential of modulating GCase activity to attenuate synucleinopathies in PD. We further validated if ABX similarly induced GCase activity in a parkinsonian LRRK2 R1441G mutant cell model. First, the basal cellular GCase activity was compared between WT and LRRK2 R1441G mutant MEFs under normal culture condition. The gene mutation effect was evident by a significant 40.4% decrease in the GCase activity in LRRK2 R1441G mutant MEFs compared to WT control (p<0.01; N=6), indicating GCase deficiency in the mutant. ABX treatment (50 μM) for 24 hr significantly attenuated such GCase deficiency in LRRK2 R1441G mutant MEFs by 21.6% increase in activity compared to DMSO-treated mutant controls (Fig. 2a) (p<0.05; N=6). Given that the GCase deficiency in LRRK2 R1441G mutant MEFs was significantly attenuated by ABX, we further investigated the effect of ABX on suppressing pSer129 in LRRK2 R1441G mutant MEFs which we engineered to stably overexpress mouse α-syn 24 . These mutant MEFs were derived from our LRRK2 R1441G mutant mice showing age-dependent accumulation of α-syn oligomers in their brains 24,25 . To address ABX drug effects, α-syn-overexpressing LRRK2 R1441G mutant MEFs were treated with ABX at 50µM for 24 hr before subjected to GCase activity assay and Western blotting ( Fig. 2c ). GCase activity assay showed that ABX treatment significantly increased GCase activity in LRRK2 R1441G mutant MEFs by 76.98% compared to DMSO-treated control cells (p<0.01, N=4) ( Fig. 2d ). Similar to the observation in SH-SY5Y cells, ABX treatment also significant reduced level of Ser129-α-syn phosphorylation by 84.33% compared to the DMSO-treated control cells (p<0.01, N=4). ABX is thermally stable The rationale of testing the thermal stability of ABX was because we aimed to determine the therapeutic benefits of ABX in mice using long-term spontaneous feeding instead of multiple oral gavages to avoid unnecessary trauma. During the drug regimen preparation, pure ABX powder was mixed with standard rodent food pellets and re-molded which involved a drying step at 70°C for 30 min. in oven. Thus, we determined the thermal stability of ABX efficacy in activating GCase activity and suppression of Ser129-α-syn phosphorylation after heat shock in oven. The stock ABX solution dissolved in DMSO (50mM) was heated at 80°C for 30 min. before cooled down and subjected to MEF treatment. Similar to the ABX without heat shock, heated ABX retained similar activation of GCase activity (77.20%, p<0.01, N=4) and reduction of Ser129-α-syn phosphorylation (70.56%, p<0.01, N=4), compared to DMSO-treated controls ( Fig. 2b-e ), indicating that the efficacy of ABX is not affected by heat. The level of GCase activation by heat-treated ABX was comparable to the levels of ABX without heat treatment. Impaired lysosomal degradation in LRRK2 R1441G mutant MEFs was attenuated by ABX treatment Studies showed that α-syn aggregates are predominantly metabolized by lysosomes 26,27 , we further determined whether increasing GCase activity by ABX treatment modulated lysosomal protease activity. First, a real-time flow cytometry assay was developed to compare lysosomal protease activity in WT and LRRK2 R1441G mutant cells using a self-quenched fluorescent substrate, DQ-BSA. Multiple clones of LRRK2 R1441G mutant MEFs were used to avoid confounding clonal effects. After ABX treatment, the intensity of emitted fluorescence upon DQ-BSA degradation in lysosomes was measured at different time points within 60 min to generate a time-dependent increase in fluorescence readouts ( Fig. 3a ). Our results showed that total lysosomal activity in mutant LRRK2 MEFs was significantly lower than that of WT controls by 57.9% as shown by AUC analysis (p<0.05; N=4) ( Fig. 3b ). Treatment of ABX (50µM) significantly attenuated the impaired lysosomal activity by increasing lysosomal activity in LRRK2 R1441G mutant MEFs by 30.4% (p<0.001; N=6). In contrast, similar ABX treatment in WT MEFs did not cause a significant effect on lysosomal activity ( Fig. 3c ). Brain penetration of ABX and drug clearance Having our in vitro findings showing ABX effects on GCase activity, we further investigated the drug benefits in our parkinsonian LRRK2 mutant mice. To avoid confounding effects of trauma from multiple oral gavages, we determined the therapeutic benefits of ABX in mice using long-term dose-controlled spontaneous feeding approach. Before long-term treatment, we determined whether oral ABX can achieve effective drug delivery to the mouse brain. First, we determined the maximum ABX concentration using LC-MS/MS achievable in serum, brain, and liver after a single oral gavage of ABX at 0.4 mg/g body weight (dissolved in physiological saline), which resulted in a peak concentration of ABX at 14.15±6.63µg/g, 199.78±117µg/g, and 123.05±35.96µg/g tissue in serum, brain, and liver, respectively, at 4 hr after oral gavage (p<0.05; N=3). The ABX level in brain, liver and serum subsequently declined to undetectable level at 48 hr post treatment ( Fig. 4a ). Next, we tested for the availability of ABX using spontaneous feeding for 14 days. Aged (14-month-old) WT and LRRK2 R1441G mutant mice were fed ad libitum with ABX-containing food pellets (3.47mg ABX/g food powder) for 14 consecutive days. On the last day of ABX treatment, the mice were euthanized before brain, liver and serum were collected for ABX quantification by LC-MS/MS. Our results showed that ABX was detected in both brain and liver after 14-day ABX treatment protocol ( Fig. 4b ). It is noteworthy that the average ABX concentration in the brain of LRRK2 mutant mice (28.08±5.9μg/g) was significantly higher than the level in WT mice (12.3±2.2μg/g) (p<0.05; N=6). However, no statistically significant difference was observed in both liver (WT: 16.86±2.39μg/g; R1441G: 21.89±7.19μg/g) and serum (WT: 1.26±0.46μg/g; R1441G: 1.47±0.38μg/g) between WT and LRRK2 mutant mice after the 14-day ABX treatment. Long-term (18-week) spontaneous feeding of ABX reduced cumulative food consumption without significant changes in average body weight To assess the long-term therapeutic effects of ABX, aged (14-month-old) WT and LRRK2 R1441G mutant mice (N≥20 animals per treatment group) were fed with food pellets infused with known amount of ABX (3.47mg ABX/g food powder) for 18 weeks. Total food consumption of each mouse was recorded per week for estimation of weekly ABX intake per mouse. The body weight of individual mouse was recorded and plotted over the course of treatment. Both WT and LRRK2 R1441G mutant mice maintained a consistent body weight over 18-week treatment ( Fig. 5a ). Also, ABX treatment did not cause significant difference in body weight in both WT and LRRK2 R1441G mutant mice compared to their respective controls without ABX. The weekly food consumption of ABX treatment in WT and LRRK2 R1441G mutant mice showed no significant difference and maintained consistently over 18 weeks of spontaneous feeding ( Fig. 5b ) Two-way ANOVA showed that LRRK2 mutation did not cause any significant effect on cumulative food consumption over 18-week of treatment. However, a significant effect of ABX treatment was observed on cumulative food consumption [F (1,32) = 20.56; p<0.001] as indicated by a lower cumulative food consumption in both WT and LRRK2 R1441G mutant mice ( Fig. 5c ). Post-hoc comparisons showed that ABX treatment significantly reduced food consumption in LRRK2 R1441G mutant mice (p<0.01; N=20), but not in WT mice ( Fig. 5c ). Based on the amount of weekly food consumption in each mouse, the average weekly dose of ABX (mg/kg/week) in 18-week treatment was calculated, showing a similar level of average weekly dosage of 325.8±23.68 and 321.7±26.50 mg/kg/week in WT and LRRK2 mutant mice, respectively (N≥20) over the whole course of treatment ( Fig. 5d ). Long-term spontaneous feeding of ABX increased GCase activity in striatum without affecting GBA protein levels After we confirmed the long-term feeding protocol, we investigated whether ABX treatment can induce GCase activity in striatum, the most susceptible brain region to synaptic dysfunction and neurodegeneration in early PD. The relationship of ABX and brain GBA protein level was investigated since GBA gene encoded for GCase. GCase activity assay showed that striatal GCase activity of LRRK2 R1441G mutant mice was significantly lower than that of WT controls by 13.1% (p<0.05; N³16). However, the total cellular GBA level in striatum did not show significant difference between these two mouse lines ( Fig. 6a ). More importantly, 18-week ABX treatment significantly increased GCase activity in WT by 21.6% (p<0.05; N≥17) and LRRK2 R1441G mutant mice by 40.6% (p<0.01; N≥16) ( Fig. 6a ). ABX treatment did not affect total GBA level in striatum of both WT and mutant mice ( Fig. 6b ), indicating that ABX-induced GCase activation was not due to increased protein expression. Long-term spontaneous feeding of ABX did not affect Ser129 - α-syn phosphorylation levels in striatum Phosphorylation of α-syn at residue Ser129 (pSer129) has been considered a surrogate marker of synucleinopathies in PD 23 . To explore whether ABX-induced GCase activation modulated α-syn phosphorylation, we assessed the levels of pSer129-α-syn in striatum by western blotting in WT and LRRK2 mutant mice after 18-week ABX feeding. Western blot analysis showed that the basal level of pSer129 in striatum were similar in WT and LRRK2 mutant mice (N≥16) ( Fig. 7a ). The total α-syn level as determined by ELISA also showed no statistically significant difference between the two mouse lines (N≥16). Spontaneous feeding of ABX over 18 weeks also did not cause any significant effects on pSer129 level in striatum of both WT (N≥17) and mutant mice (N≥16) ( Fig. 7b ). This in vivo observation contrasted the effects of ABX which significantly suppressed Ser129 phosphorylation seen in SH-SY5Y and MEFs ( Fig. 1 & Fig. 2 ). Long term spontaneous feeding of ABX reduced soluble α-syn oligomer levels in mutant LRRK2 R1441G mouse striatum Pathogenic LRRK2 R1441G mutation has been associated with increased accumulation of α-syn oligomers in the brain 24 . Although ABX treatment did not show a significant effect on striatal Ser129-α-syn phosphorylation, we further investigated the therapeutic effects on α-syn oligomer level in the brain. α-syn oligomers in cortex and striatum of ABX treated mice were quantified using a validated, conformational-specific ELISA. Briefly, brain tissues were freshly lysed by sonication in detergent-free PBS supplemented with protease inhibitors to extract the total cell lysates containing soluble α-syn oligomers. Total oligomer levels (pg/mg total protein) in target brain regions were quantified based on linear standard curve provided by the kit, and normalized by mg total cellular protein. Our ELISA results showed that total soluble α-syn oligomers levels in both striatum and cortex were significantly higher in untreated LRRK2 R1441G mutant mice compared WT control mice (79.7%, 20.6%; p<0.01; N≥16) ( Fig. 8 ), consistent with our earlier findings 24 . Following 18-week spontaneous feeding of ABX, striatal α-syn oligomer levels in ABX-treated mutant mice were significantly reduced (23.4%; p<0.05; N≥16), indicating that our long-term treatment regimen was efficacious in reducing abnormal accumulation of α-syn oligomer levels in mutant to normal physiological levels comparable to those in WT mice ( Fig. 8a ). Unlike the effects in the striatum, long-term ABX treatment did not reduce cortical oligomer levels in mutant LRRK2 mice ( Fig. 8b ). Discussion The correlation between GCase dysfunction and PD pathogenesis is increasingly recognized as a critical factor underlying nigrostriatal dopaminergic neurodegeneration. Mutations in the GBA1 gene, which encodes GCase, constitute the most significant known genetic risk factor for the development of PD. Clinical studies showed that GCase expression and activity are both decreased in idiopathic PD brain regions (e.g. caudate and substantia nigra) where they accumulate misfolded α-syn protein 19 . This relationship is underscored by findings showing that GCase deficiency enhances α-syn aggregation in PD 28 . Our current findings consolidated this pathological relationship between GCase activity and synucleinopathies in the context of PD, particularly on the role of soluble oligomeric forms of α-syn. In our study, we observed that the lysosomal function and GCase activity were reduced in LRRK2 R1441G mutant MEFs and mice striatum compared to their WT littermates, reflecting similar clinical situations in human PD. Our results demonstrated that GCase activity and lysosomal function were significantly enhanced following ABX administration in both in vitro cell models (human SH-SY5Y and MEFs) and in aged LRRK2 R1441G mutant mice. Previously, we elucidated an age-dependent accumulation of α-syn oligomers in LRRK2 R1441G mutant mouse striatum, which mirrors key prodromal phenotypic changes of PD. This age-dependent phenomenon began in mutant mice at 14 months of age, with oligomer levels rising progressively by 18 months 24 . To test our hypothesis whether activation of GCase can modulate α-syn oligomer level in the brain, an 18-week spontaneous feeding regimen of ABX was given to 14-month-old LRRK2 R1441G mutant mice to induce GCase activity and lysosomal function. Our treatment protocol achieved an average weekly dose at 300mg/kg of ABX based on individual mouse weekly food consumption. This dosage level is much lower than the reference LD 50 of ABX administered orally in mice at 2380 mg/kg ( Registry of Toxic Effects of Chemical Substances (RTECS), The Dictionary of Substances and their Effects, 1st Edition, IUCLID ). Our current dose showed significant increase of GCase activity in the mutant mouse brains, where the α-syn oligomer level was concomitantly reduced. Given that higher-ordered α-syn aggregates are predominantly metabolized via autophagic lysosomal degradation 29 , this is the first study to demonstrate that long-term therapeutic activation of GCase and lysosomal activity can attenuate accumulation of toxic α-syn oligomers in aged brains. Our findings also reiterate the role of soluble α-syn oligomers as a modifiable therapeutic target to attenuate synucleinopathies in PD. As a proof concept, we utilized ABX as a known therapeutic GCase activator for cell and mouse treatment. ABX is a known drug that can break up phlegm in the treatment of respiratory diseases associated with viscid or excessive mucus, e.g., bronchitis or asthma. Here, we tested whether long-term feeding of ABX could maintain an elevated level of brain GCase activity and showed beneficial effects to reduce α-syn oligomer accumulation in our aged LRRK2 mutant mouse brains. We first considered the route of drug administration by spontaneous feeding which would not cause trauma or stress to the testing animals over a long period of treatment time. We prepared the mouse feed with a known amount of ABX based on an estimation of 7 grams of daily food pellet consumption per mouse (C57BL/6 mouse). After 18-week of spontaneous feeding, the average body weight of the treated mice showed no significant difference compared to untreated mice. This indicated that our feeding protocol is feasible to control ABX dosage and avoid unnecessary trauma caused otherwise by multiple daily oral gavages over 18-week. It is noteworthy that each mouse consumed a slightly different amount of food per day. However, since ABX was fed over a long period of time, the average weekly dose of ABX in both WT and LRRK2 R1441G mutant mice were comparable without significant differences. This average weekly dose level of ABX achieved significantly increased GCase activity in the brain after 18-week treatment regimen. Although the molecular link between GCase activity and α-syn accumulation in PD brain is still unclear, our data supported our hypothesis that the observed reduction in α-syn oligomer levels in ABX-treated mouse brains can be attributed to the increased therapeutic activation of GCase, which may subsequently facilitate degradation of α-syn aggregates through improved lysosomal function. Previous studies linked GCase dysfunction with α-syn accumulation by showing that viral-mediated suppression of GCase activity in Gaucher’s disease mice led to an increase in α-syn pathology 30,31 . Conversely, activation of GCase has been shown to improve the clearance of pathological α-syn, which supports the idea of therapy targeting the accumulation of toxic species attributed to PD pathology 32 . These outcomes underscore the validity of GCase as a therapeutic target, particularly in the context of genetic variants such as LRRK2 mutations, which are associated with predisposed dysregulation of α-syn pathways. Hyper-phosphorylated α-syn is one of the major components found in the Lewy body of PD brains 33 . Phosphorylated Ser129-α-syn is a proposed surrogate marker for PD diagnosis. Abnormal phosphorylation of the protein has been shown to increase the propensity of its oligomer formation in vitro 34 . However, the level of pSer129-α-syn in striatum of our aged LRRK2 mutant mice did not exhibit significant changes after 18-week ABX treatment, yet the oligomer α-syn level was reduced by a significant 23%. This finding suggests that while GCase activation effectively reduced the accumulation of α-syn oligomers, it did not significantly impact the phosphorylation status of Ser129-α-syn in the brain. The reduction in oligomer levels without a change in phosphorylation suggests that the GCase activation may promote the clearance or disaggregation of α-syn oligomers rather than inhibited oligomer formation. This argument is supported by the significant increase of lysosomal activity in LRRK2 mutant MEFs following ABX treatment. Moreover, our previous studies elucidated lysosomal dysfunction in LRRK2 R1441G mutant mice. It is plausible that therapeutic activation of GCase enhanced lysosomal activity that facilitated more efficient degradation of α-syn oligomers via activating the lysosomal autophagic pathways in these mutant animals. Furthermore, since glucosylceramide (GlcCer) accumulation is known to promote α-syn aggregation by stabilizing α-syn oligomers 35 , increasing GCase activity could reduce GlcCer level that subsequently destabilized existing aggregates for downstream lysosomal degradation. The lack of change in pSer129-α-syn levels suggests the ABX intervention preferentially targeted oligomer clearance rather than modulating phosphorylation-dependent aggregation of α-syn. Improving GCase function while controlling α-syn oligomer levels appears to be a sensible approach to delay neurodegenerative processes linked with PD. Recent studies focused on improving GCase activity as the therapeutic strategies by using the GCase activator compounds including ABX. Supportive to our findings, these compounds improved GCase activity in neurons derived from PD patients with GBA mutations 30 . Besides ABX, a study applied small molecule S-181 as the GCase modulator on iPSC-derived dopaminergic neurons from PD patients with GBA1 mutation and GBA1 mutant mice to enhance GCase activity. The results showed that the GCase activity was increased while the accumulation of α-syn was reduced in these patient neurons in vitro 36 . The current landscape of clinical research underscores the potential of GCase activators as disease-modifying therapies for PD by improving lysosomal function and mitigating neurodegenerative changes induced by α-syn accumulation. Currently, clinical studies are underway using GCase activator for the treatment of PD, mainly in patients carrying GBA1 mutation. For instance, Vanqua Bio announced positive interim results from phase 1 clinical trial of VQ-101, an orally administered, brain-penetrant, allosteric activator of GCase for the treatment of GBA-PD 37 , which aimed to reduce α-syn accumulation. These studies highlight the growing interest in GCase activators to target the underlying lysosomal dysfunction in PD. Our current findings highlight the benefits of chronic therapeutic GCase activation to reduce progressive accumulation of α-syn oligomers in LRRK2-associated PD. Such pathology was confidently shown in PD patients by two recent independent clinical studies 17,18 . In conclusion, our study demonstrated for the first time a feasible therapeutic approach to induce GCase activity, showing a concomitant reduction of α-syn oligomer accumulation in the brains. Our aged LRRK2 R1441G mutant mice are well-characterized disease models demonstrating progressive accumulation of toxic soluble α-syn oligomers in the brains with age. This model is valuable to reveal molecular events at the prodromal stage of PD for testing early therapeutic options. The therapeutic outcomes from long-term spontaneous feeding of GCase activator in our mutant mice shed light on new strategies to attenuate synucleinopathies by inducing GCase activity and lysosomal degradation of toxic oligomers in PD brains. Although ABX has well-documented drug safety profile in human as medicine, further investigation using other brain-penetrant GCase activators is needed to confirm the specific effect of GCase activation and its modulation on α-syn pathologies. Future investigations include elucidation of the mechanism(s) regarding the combined effects of GCase activation and α-syn post-translational modifications. By addressing these disease-related pathways, we may enhance the capacity to alter disease progression related to synucleinopathies and potentially improve clinical outcomes for PD. Our recent report on therapeutic LRRK2 inhibition is a vivid example to attenuate aberrant LRRK2 kinase hyperactivity that modulated cellular clearance of α-syn oligomers 22 . This could be a novel approach by combining GCase activator and LRRK2 inhibitor that ameliorates aberrant LRRK2 kinase hyperactivity and reduces the accumulation of α-syn oligomers, the emerging toxic species that were previously underestimated in PD pathogenesis and treatment. Methods LRRK2 R1441G homozygous knockin mouse model A C57BL/6 mice with complete homozygous knockin of pathogenic LRRK2 R1441G mutation (cDNA 4321C>G) maintained under pure C57BL/6N mouse background 38 . All mice were housed in the Laboratory Animal Unit, HKU with accreditation through the Association for Assessment and Accreditation of Laboratory Animal care international (AAALAC), under standard conditions (12-h light/dark cycle) with unrestricted access to food and water. The procedure of experimental use of animals was approved by the Institutional Animal Care and Use Committee, HKU (CULATR#4506-17). All studies involved followed the ARRIVE guidelines. Genotype of animals was determined by DNA sequencing. Mouse embryonic fibroblasts (MEFs) culture Homozygous LRRK2 R1441G knockin mutant mice carrying mutation (cDNA 4321 C>G) of LRRK2 and their wild-type littermates were used for preparation of mouse embryonic fibroblasts (MEFs) according to our published protocol 24 . Individual mouse embryos at day E12.5 were isolated from crosses between two heterozygous LRRK2 WT/R1441G mice 24 . Individual clone of MEFs was obtained from each embryo and genotyped at passage 1 when developed into proliferating culture. All MEFs were cultured in Dulbecco’s Modified Eagle medium (DMEM; ThermoFisher™ Scientific, 10,569–010) containing 15% Fetal Bovine Serum (FBS; GE Healthcare HyClone™, SH30071.03), 100 units/ml penicillin, 100 μg/mL streptomycin (ThermoFisher™ Scientific, 15,140–122), non-essential amino acids ThermoFisher™ Scientific, 11,140–050). Stable overexpression of human α-syn in SH-SY5Y cell line To assess the effect of GCase activation on α-syn-Ser129 phosphorylation, human α-syn protein was stably overexpressed in SH-SY5Y cells (ATCC; CRL-2266) by transduction of lentivirus following our previous published protocol 22 . All SH-SY5Y cell lines were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, ThermoFisherTM Scientific, 11320-082), supplemented with 10% fetal bovine serum (FBS; GE Healthcare HyCloneTM, SH30071.03), 1% 100 units/ml penicillin, and 100 μg/mL streptomycin (Gibco, ThermoFisherTM Scientific, 15140–122). To generate α-syn expression construct, human SNCA (NCBI Entrez Gene: 6622) gene was amplified by PCR from the total cDNA of SH-SY5Y cells as a template to generate an insert of cDNA fragment encoding for h SNCA. This h SNCA cDNA insert was then sub-cloned into a lentiviral backbone plasmid pSIN4-EF2-IRES-Pur derived from a gift (Addgene™ plasmid, 16580) to construct pSIN4- h SNCA plasmid (Addgene™ plasmid, 102366). Expression levels of total and phosphorylated Ser129-α-syn (pS129) were determined by Western blotting. Cells at 80% confluence were treated with a GCase activator, Ambroxol™ (ABX; A9797; Sigma Aldrich), at 10 and 50µM for 72 hr before GCase activity assay and Western blotting. Negative control cells were treated with DMSO (v/v 0.01%). Feeding of ABX by food pellets in mice To achieve long-term feeding of ABX without traumatizing the mice, ABX was mixed into food pellets for ad libitum feeding. Normal mouse diet (150g; PicoLab® Rodent Diet 20, LabDiet™, Cat. no 5053) was grinded into powder and then mixed with 520mg of ABX aiming to achieve the dose of 800mg/kg per day [the highest reported dose without noted toxic effect 39,40 ] based on estimated daily consumption of 7 grams of food pellet per C57BL/6 mouse. 150ml of sterilized distilled water was added into the mixture of ABX and ground food powder and moulded into size similar to normal food pellet. The ABX-containing food pellets were dried at 70°C in oven overnight and stored at room temperature. Treatment of ABX in cell culture To induce GCase activity, ABX was dissolved in dimethyl sulfoxide (DMSO) to produce a 50mM stock solution. ABX treatments and vehicle (DMSO) controls were done to achieve a 0.01% DMSO concentration (v/v) in culture medium. Unless otherwise specified, dissolved ABX remained in the culture medium until time of harvest without medium replacement. Cell Harvest and Immunoblotting Treated cells (both SH-SY5Y and MEFs) were harvested through washing with culture medium and collection of a cell pellet through centrifugation at 4°C for 10 min at 2500 rpm. The medium was aspirated, and cell pellet was resuspended in 1x RIPA buffer without SDS (Cell Signaling Technology, 9803S) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail, 100x (ThermoFisher Scientific, 78446) and 2% phenylmethylsulfonyl fluoride (PMSF) (Pierce, 36978). Protein lysates were incubated on ice for 30 min, followed by centrifugation at 4°C for 15 min at 14000 rpm. Protein concentration was determined through Bradford Assay (Quick Start™ Bradford 1x Dye Reagent, #5000205). Equal amounts of proteins were dissolved in sample buffer (Thermo Scientific™ Pierce™ Lane Marker Non-Reducing Sample Buffer, 39001), and electrophorized in 4% stacking, 12% resolving SDS-polyacrylamide gels, at 80V. The resulting gels were electro-transferred to PVDF membranes. The resulting membranes incubated in 4% paraformaldehyde solution in PBS (Santa Cruz Biotechnology, 30525-89-4) for 20 min, and was subsequently blocked in 5% bovine serum albumin (Sigma Aldrich, A9647) in TBST (Santa Cruz Biotechnology – sc362311), and probed with primary antibodies overnight and HRP-conjugated secondary antibodies (P0260, Polyclonal rabbit anti-mouse immunoglobulins/HRP, Agilent DAKO™; or P0448, Goat Anti-Rabbit Immunoglobulins/HRP Agilent DAKO™) for one hour in 1% bovine serum albumin in TBST. This was followed by incubation in ECL substrate solution for chemiluminescence visualization. The quantification of band intensity was done using the Image Lab Software (BioRad). Lysosomal activity assay A real-time flow cytometry assay was developed to compare lysosomal protease activity in WT and LRRK2 mutant cells. Multiple clones of LRRK2 mutant MEFs were used to avoid confounding clonal effects. Cells in refreshed medium for 2 hr were treated with a lysosome protease substrate, DQ-BSA (10 µg/ml), for different time points. Intact DQ-BSA protein does not emit fluorescence 41 . However, once this protein substrate is degraded by protease in lysosomes, it is broken down into protein fragments with isolated fluorophores (red). After such de-quenching by DQ-BSA degradation in lysosomes, the fluorescent intensity in cells was measured by flow cytometry (excitation: 590 nm; emission: 620 nm). The increased amount of fluorescent DQ-BSA metabolites as calculated by the area-under-curve (AUC) analysis reflected the level of lysosomal activity. GCase enzymatic activity assay for mouse brain tissues and cell lysates Mouse brain and peripheral organ tissue samples were homogenized in cold PBS buffer by sonication with 1X protease inhibitor cocktail (ThermoFisher Scientific, 186284), 1X EDTA (ThermoFisher Scientific, 1861283) and 1mM phenylmethylsulfonyl fluoride (PMSF) (Pierce, 36978). Cell lysates were homogenized in 1X RIPA buffer without SDS (Cell Signaling Technology, 9803S) supplemented with 1X protease inhibitor cocktail and 2X PMSF. Homogenates were centrifuged at 4 o C for 10 min at 14000rpm and protein concentration of the supernatant was measured using Bradford Assay (BioRad, Quick Start™ Bradford Protein Assay, 5000205). Resulting lysate was diluted to 1µg/µl in McIlvaine buffer (pH 5.4, mixing of 0.1M citric acid (Sigma-Aldrich, 251275) and 0.2M disodium phosphate (Sigma-Aldrich, S-0876). GCase activity was measured by incubating 20µl of diluted lysate with 5mM 4-methylumbelliferyl-beta-d-glucopyranoside (Sigma-Aldrich, M3633) in 40µl McIlvaine buffer supplemented with 22mM sodium taurocholate hydrate (Sigma-Aldrich, 86339) at room temperature for 3 h (brain or peripheral organ samples) or for 30 min (cell lysates) 42 .Standard curve was generated from 250 µM to 1.95 µM of 4-methylumbelliferone (4-MU) (Sigma-Aldrich, M1381) dissolved in DMSO. The reaction was terminated by adding 50 µl of 1 M glycine buffer (Affymetrix, Glycine, 16407 5KG) (pH 10.0) which is dissolved in water, and the fluorescence from cleaved product (4-MU) was measured using a spectrophotometer (Ex 365 /Em 450 ). All the samples were done in duplicate. GBA protein expression level in mouse brain tissues Total glucosylceramidase (GBA) level in mouse brain was quantified using a commercial ELISA kit according to manufacturer’s protocol (MyBiosource.com, MBS7206378). Protein concentration of brain lysates was determined using Bradford assay. Standards were provided from the kit and diluted samples were added into anti-GBA antibody coated wells. The optical density (OD) at 450nm was measured by a microplate reader. The concentration of GBA was calculated by a four-parameter logistic (4-PL) curve-fit or logit-log linear regression curve. Tissue extraction and quantification of ABX using LC-MS/MS To determine the bioavailability of ABX, we quantified the drug in mouse serum, liver and brain using LC-MS-MS. Briefly, ABX in serum, liver and brain samples was extracted by sonicating the homogenized samples for 20, 40 and 40 min with 4 ml of acetonitrile, respectively. The samples were then centrifuged at 10,000 rpm for 10 min to remove insoluble tissue debris. The supernatant was deproteinized and delipidated twice by the addition of 2 ml of acetonitrile and hexane, respectively. The clean acetonitrile layer was obtained after centrifugation at 10,000 rpm for 10 min. The clean acetonitrile layer was then collected for future analysis. An Agilent (Palo Alto, CA) 1290 Infinity liquid chromatograph coupled to a SCIEX QTRAP 3200 tandem mass spectrometer (Woodlands, Singapore) with an electrospray ionization interface was used. The analytical column is ZORBAX Eclipse Plus C18 (2.1 × 100 mm, 1.8 µm, Agilent) equipped with its corresponding guard column (5 × 2.1 mm, 1.8 µm, Agilent). The elution was conducted under isocratic conditions with a mobile phase composed of 90% methanol (with 10 mM ammonium acetate) and 10% Milli-Q water. Quantification of total and oligomeric α-syn level The oligomeric α-syn level in the mouse brain lysates after 18-week ABX ad libitum feeding were quantified using a commercial ELISA kit with assay sensitivity of 1.0pg/ml (MBS724099; MyBioSource.com™) according to our published protocol 22 . Mouse brain tissues were freshly homogenized in ice-cooled PBS supplemented with Halt™ Protease and Phosphatase Inhibitor (ThermoFisher™ Scientific, 78444), PMSF (Pierce™, #36978) and EDTA to preserve the native α-syn oligomer. Homogenates were briefly centrifuged for 5 min at 720 × g to remove fatty tissues and nuclei. Resultant supernatants containing cytoplasmic fraction and small cellular organelles were further clarified by centrifugation at 4 °C for 15 min at 12,000 × g. Cytosolic fractions containing soluble α-syn oligomers were collected from the resultant supernatants. Protein concentrations of PBS-soluble lysates were determined by Bradford assay (ThermoFisher™ Scientific, #5000205). Briefly, 500–700 μg of total lysates were subjected to α-syn oligomer ELISA. Quantification of α-syn oligomer levels in PBS-soluble lysates (pg oligomers/mg total protein) was based on the linear standard curve generated from recombinant α-syn oligomer standards provided by the kit. Total α-syn was quantified by another commercial ELISA kit (ThermoFisher Scientific™; KHB0061) according to manufacturer’s protocol. Statistical analysis All experiments were performed based on independent trials to achieve statistical significance, as indicated in figure legends. Results were expressed as means ± SEM. Conclusions were drawn based on statistical analyses using GraphPad™ PRISM software (GraphPad Inc., CA). The normality of data sets was determined using D’Agostino & Pearson omnibus normality test. Potential outliers were identified using Grubb’s test. LRRK2 mutation effect and differences between the ABX-treatment and untreated control groups were determined by unpaired Student’s t-test to demonstrate drug effect. Alternative analysis was performed using non-parametric, Mann–Whitney U-test to compare differences between two independent groups when the dependent variables did not fulfill data normality assumptions. Group comparisons were considered significant when p-value was less than 0.05 (p < 0.05). Declarations Data availability There are no data in this paper that can be submitted to a data repository. The data that support the findings in this article are available from the corresponding author on request. Acknowledgements We would like to acknowledge Tai Hung Fai Charitable Foundation - Edwin S H Leong Research Programme for Parkinson’s Disease (PWL Ho; #P0054772) for their long-term trust and funding support. We also acknowledge funding support by Health and Medical Research Fund (HMRF; #07183516), Health Bureau, Hong Kong SAR, China. Postdoctoral Fellowship (E.E.S. Chang) was supported by SKLMP Seed Collaborative Research Fund, State Key Laboratory of Marine Pollution, City University of Hong Kong (CityU), and Postdoc Matching Fund Scheme 23/24 (P0050969), The Hong Kong Polytechnic University (HK PolyU; Fund holder: P.W.L.H.). Research consumables and staff cost were partly supported by Start-up Fund for New Recruit, HK PolyU (Fund holder: P.W.L.H.), and an internal department fund by Department of Rehabilitation Sciences, HK PolyU (Fund holder: Benjamin K. Yee, Co-I: P.W.L.H.). Our appreciation of technical assistance by Dr. Phoebe Ruan, and Qi Wang from CityU on LC-MS/MS measurements and data analysis. The authors also thank for the administrative supports from the Department of Rehabilitation Sciences, HK PolyU, and the Department of Medicine, The University of Hong Kong. Author contributions P.W.L.H., Z.Y.K.C., H.L., and S.L.H. designed the project. Z.Y.K.C., H.L., E.E.S.C. P.R., Q.W., I.L.L., Y.M. and S.X.Y.Z performed experiments and data analysis. Z.Y.K.C. H.F.L. and B.W.M.L. managed laboratory logistics and safety. P.W.L.H. and S.Y.Y.P. oversaw and evaluated all experimental data. Z.Y.K.C., P.W.L.H. and S.Y.Y.P. wrote and review the manuscript with input of all co-authors. All authors read and approved the final manuscript. Competing interests: The authors declare no competing interests, and all authors have agreed to publish this article. References Abdullah R. , et al. Parkinson's disease and age: The obvious but largely unexplored link. 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10:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7185990/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7185990/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41531-025-01205-7","type":"published","date":"2025-12-12T15:59:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87812113,"identity":"c33bbe6f-f413-41c9-a882-c5599a8fa4ff","added_by":"auto","created_at":"2025-07-29 09:33:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2704095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmbroxol treatment increased glucocerebrosidase (GCase) activity in human SH-SY5Y neuroblastoma cell lines – Normal SH-SY5Y, and SH-SY5Y stably overexpressing human α-syn\u003c/strong\u003e \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eh\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eSCNA).\u003c/strong\u003e (a) Cells were refreshed with new medium for 2 hrs before Ambroxol treatment for an additional 72 hrs. (b) GCase activity was measured using the 4-MU fluorescence assay, and a consistent dose dependent increase was observed both normal and α-syn overexpressing cells at 50µM Ambroxol treatment (p\u0026lt;0.01; N=7). (c) Western blots showing the effects of Ambroxol on GBA, total and phosphorylated Ser129 α-syn expression. Total cellular GBA protein level was significantly increased in both normal and α-syn overexpressed SH-SY5Y cells (p\u0026lt;0.01 and p\u0026lt;0.05, respectively). The levels of pSer129 α-syn was decreased by 16.9% decrease with treatment of 50µM Ambroxol (p\u0026lt;0.05). However, there was no significant changes in the total levels of α-synuclein with Ambroxol treatment. Data represents mean ± standard error of mean (SEM) from seven independent experiments (N=7). Comparison between different Ambroxol concentrations in same cell line was analyzed through 2-way ANOVA multiple comparisons with post-hoc Tukey’s test. Statistical significance between cell lines was analyzed by unpaired Student’s t-test. *p\u0026lt;0.05, **p\u0026lt;0.01 represent statistical significance between two designated groups.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/b2a48c788ef6c4c1d810d6c6.png"},{"id":87813660,"identity":"265a3ed7-d9c4-40f2-b672-6e86f21da96b","added_by":"auto","created_at":"2025-07-29 09:41:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1488360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmbroxol (ABX) enhances GCase activity and reduces Ser129-α-syn phosphorylation in LRRK2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eR1441G\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutant MEFs with and without thermal processing. \u003c/strong\u003e(a) A significant reduction in cellular GCase activity was observed in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs (R1441G) compared to wild-type (WT) MEFs (p\u0026lt;0.01; N=6), indicating GCase deficiency. Following the treatment of 50µM ABX, GCase activity in mutant MEFs was significantly increased compared to untreated controls (p\u0026lt;0.05), suggesting that ABX treatment attenuated GCase deficiency caused by the LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutation. (b) The stock ABX solution dissolved in DMSO (50mM) was heated at 80°C for 30 min. before subjected to MEF treatment in parallel with ABX without heat treatment. (c) Representative Western blots showing changes in expression level of total and pSer129-α-syn and GBA proteins after ABX treatment. (d-e) Changes in GCase activity and pSer-129/total α-syn after ABX treatment. Data represents mean ± standard error of mean (SEM) from four independent treatments (N=4). Statistical significance was analyzed by one-way ANOVA with post hoc Tukey’s multiple comparison correction.\u0026nbsp; *p\u0026lt;0.05, **p\u0026lt;0.01 represent statistical significance between two designated groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/573ee6adb26281ad9d184b87.png"},{"id":87811557,"identity":"249be13c-cd6f-4a88-8f55-ae8c375d4d88","added_by":"auto","created_at":"2025-07-29 09:25:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1448739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmbroxol treatment ameliorated lysosomal dysfunction in LRRK2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eR1441G\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutant MEFs.\u003c/strong\u003e (a) Development of lysosomal degradation assay using DQ-BSA (lysosomal substrate) using flow cytometry. A time-dependent increase in fluorescence intensity as measured by flow cytometry. The rate of increase in fluorescence reflected the rate of lysosomal (protease) degradation. \u0026nbsp;(b) LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant cells showed significantly lower lysosomal activity than WT control cells based on DQ-BSA lysosomal protease activity assay. (WT: Wildtype; KI: LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant). (c) Treatment of ABX significantly increased DQ-BSA lysosomal activity in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs. Data represents mean ± standard error of mean (SEM) from six independent experiments (N=6). Statistical significance was analyzed by unpaired Student’s t-test.\u0026nbsp; *p\u0026lt;0.05, ***p\u0026lt;0.001 represent statistical significance between two designated groups. ns=not significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/30802a53548ab6bddf7cc726.png"},{"id":87813661,"identity":"9a90da30-00ea-4dc6-bfdf-eac8059f2fe5","added_by":"auto","created_at":"2025-07-29 09:41:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2180155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of ABX in mouse brain, liver and serum using LC-MS-MS\u003c/strong\u003e. (a) Time-dependent clearance of ABX in WT brain after a single oral administration of ABX (400 mg/kg) using LC-MS-MS. ABX concentration reached peak level at 4 hrs after single oral administration of ABX and declined gradually to 48 hrs post treatment indicating the drug has completely metabolised. (b) 14-month-old WT and LRRK2 mutant mice were fed \u003cem\u003ead libitum \u003c/em\u003ewith custom-made food pellet mixed with known amount of ABX (3.47mg ABX/g food powder) for 14 consecutive days before quantification of ABX (N=6). Results are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/d8d41e5674df3b91c44ab1f6.png"},{"id":87811563,"identity":"f19c6d9f-fd0d-483c-983b-6793ac2fb278","added_by":"auto","created_at":"2025-07-29 09:25:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3064019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime-dependent plots of average body weight (a), weekly consumption of ABX food pellet (b), cumulative food consumption (c) and weekly dose of ABX consumed over 18-week ABX treatment (d).\u003c/strong\u003e Long term \u003cem\u003ead libitum\u003c/em\u003e feeding of ABX reduced cumulative food consumption but maintained a steady average body weight during the whole course of treatment. The cumulative food consumption with ABX was significantly less than normal diet in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice while no significant difference in both normal and ABX diet in WT mice (c). The average ABX dose consumed over 18-week was no significant difference in WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (d). Data represents mean ± standard error of mean (SEM). Statistical significance was analyzed by post-hoc Tukey’s multiple comparison between two designated groups.\u0026nbsp; **p\u0026lt;0.01 represent statistical significance between two designated groups. ns=not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/1400d6b7302e0cb5ebbf1398.png"},{"id":87811558,"identity":"88b1e4ca-8c8a-46e5-a127-d33c88ce9cb5","added_by":"auto","created_at":"2025-07-29 09:25:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2124475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired GCase activity in LRRK2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eR1441G\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutant mice was attenuated by 18-week ABX treatment.\u003c/strong\u003e Striatal GCase activity in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mouse striatum was significantly lower than that in WT control mice (N≥16). After 18-week ABX treatment, the GCase activity was significantly increased in both WT control and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (a). Striatal GBA expression level was no significant differences in untreated WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice. Similarly, ABX treatment did not affect total GBA level in striatum of both WT and mutant mice (b). Data represents mean ± standard error of mean (SEM). Statistical significance was analyzed by Student’s t test between two designated groups. *p\u0026lt;0.05, **p\u0026lt;0.01 represent statistical significance between two designated groups. ns=not significant.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/4573750d0697cc1dfb91b5a6.png"},{"id":87811564,"identity":"4df6b7f0-da9b-4795-a5c4-067fb976c6ca","added_by":"auto","created_at":"2025-07-29 09:25:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2383313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhosphorylation of Ser129-α-syn (pSer129) in striatum of WT and LRRK2\u003c/strong\u003e\u003csup\u003eR1441G\u003c/sup\u003e\u003cstrong\u003e mutant mice after ABX treatment.\u003c/strong\u003e \u0026nbsp;ABX treatment did not affect both pSer129 and total α-syn level in striatum of both WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (N≥16). Data represents mean ± standard error of mean (SEM). Statistical significance was analyzed by Student’s t test between two designated groups. ns=not significant.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/bb7ce40fe1cd6dbc3757014d.png"},{"id":87814097,"identity":"e8d736b6-b6ec-43c4-b5da-cd5aa86cb138","added_by":"auto","created_at":"2025-07-29 09:49:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2123283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e18-week spontaneous feeding of ABX reduced α-syn oligomers in LRRK2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eR1441G\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutant mouse brain striatum, but not in cortex.\u003c/strong\u003e (a-b) Total soluble α-syn oligomers levels (pg/mg total protein) in both striatum and cortex were quantified by validated, conformation-specific ELISA, based on linear standard curve generated from standards provided by the kit. Assessment of soluble lysates from untreated WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice revealed significantly higher α-syn oligomers level in the mutant compared to their age-matched WT littermates. The α-syn oligomers level in 18-week ABX treated LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant brain striatum was significantly reduced compared to their untreated mutant control mice (a). Unlike striatum, the α-syn oligomers level in cortex showed no significant difference after 18-week ABX treatment. Data expressed as mean ± SEM. Datasets were subjected to D’Agostino \u0026amp; Pearson normality test prior to statistical analysis. *p \u0026lt; 0.05 and **p \u0026lt; 0.01 represent statistical significance between two designated groups by unpaired, parametric Student’s t-test. “ns” not significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/6f7800ec0659c45df0d85191.png"},{"id":98245738,"identity":"16ef6f23-0886-44f7-9e37-13db98ad1679","added_by":"auto","created_at":"2025-12-15 16:18:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20212211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7185990/v1/d5cce123-8e28-4da1-b8dc-603e5be482f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-term oral regimen of glucocerebrosidase activator reduces a-synuclein oligomer accumulation in aged LRRK2 mutant mouse brains - therapeutic implication of Parkinson’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a progressive neurodegenerative disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons, associated with characteristic motor and non-motor symptoms. The pathological deposition of misfolded alpha-synuclein (\u0026alpha;-syn) protein in the disease brain involves prior post-translational modification (e.g., phosphorylation) and progressive aggregation into toxic higher-molecular-weight soluble oligomers and insoluble fibrils (in form of Lewy bodies). These events collectively contribute to neuronal toxicities that hasten disease progression as the central hallmark of PD\u003csup\u003e1\u003c/sup\u003e. Despite that a number of genetic mutations have been definitively linked to the onset of the disease\u003csup\u003e2\u003c/sup\u003e, the common mechanism involved remains poorly understood. Current treatment options offer only symptomatic relief without halting neurodegeneration. Disease-modifying strategies to delay progression remains the most plausible approach. Recent research has identified new genetic risk factors associated with PD. The most prominent one is GBA1 (GBA; NCBI Gene ID: 2629) mutations\u003csup\u003e3\u003c/sup\u003e. Approximately 5-10% of PD patients among different populations worldwide were found to carry GBA mutations, making this gene one of the most important genetic predisposing risk factors. Glucocerebrosidase (GCase) is a lysosomal enzyme encoded in GBA1 gene which is responsible for the breakdown of glucocerebroside (GlcCer) to glucose and ceramide. GCase deficiency can lead to the accumulation of toxic glycosphingolipids which potentiate \u0026alpha;-syn aggregation with yet-unclear mechanism(s)\u003csup\u003e4,5\u003c/sup\u003e. Experimental augmentation of GCase activity in mouse neurons has been shown to modulate pathological \u0026alpha;-syn insults\u003csup\u003e6\u003c/sup\u003e. Whilst this evidence supported an association between GCase dysfunction and \u0026alpha;-syn aggregation, the beneficial effects of modulating GCase activity in PD patients await further validation. \u003c/p\u003e\n\u003cp\u003eAs a native presynaptic protein, \u0026alpha;-syn exists in a dynamic equilibrium amongst different conformations\u003csup\u003e7\u003c/sup\u003e. Growing evidence supported that the soluble oligomeric species of \u0026alpha;-syn contribute to synucleinopathies in PD\u003csup\u003e8\u003c/sup\u003e. Various detection techniques of synucleinopathies were recently developed and validated under clinical settings. For instances, \u0026alpha;-synuclein seed amplification assays (SAA), including real-time quaking-induced conversion (RT-QuIC), can now detect synucleinopathies from newly-diagnosed PD patient cerebrospinal fluid (CSF) samples with high sensitivity and specificity\u003csup\u003e9\u003c/sup\u003e. Moreover, new \u0026alpha;-syn PET tracers were introduced to visualize \u0026alpha;-syn pathologies in prodromal PD brains\u003csup\u003e10,11,12\u003c/sup\u003e. These diagnostic tools revealed persuasive clinical evidence that clarified the paradox of soluble seed-competent \u0026alpha;-syn oligomers in PD pathogenesis and progression\u003csup\u003e13\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eLeucine-rich repeat kinase-2 (LRRK2) mutations represent one of the most common genetic risks in both familial (5-13%) and sporadic (1-5%) PD. Clinically, LRRK2 mutation carriers who develop PD are largely indistinguishable from late-onset idiopathic cases\u003csup\u003e14\u003c/sup\u003e, implying that LRRK2-PD share common disease mechanisms. This protein encodes for two distinctive enzymes, a protein kinase and a putative GTPase, within a single polypeptide chain, serving as an important intracellular signal transduction protein. The LRRK2\u003csup\u003eR1441G\u003c/sup\u003e (4321C\u0026gt;G) mutation causes hyperactive kinase activity, and it adversely affects GCase activity that compromised lysosomal function and neuronal survival\u003csup\u003e15,16\u003c/sup\u003e. Two recent independent studies revealed increased \u0026alpha;-syn in postmortem brain tissue from LRRK2-associated PD, resembling oligomeric accumulations rather than typical fibrillar Lewy body inclusions\u003csup\u003e17,18\u003c/sup\u003e. Selective loss of GCase activity is associated with the aggregation of \u0026alpha;-syn and increased accumulation of \u0026alpha;-syn oligomers in sporadic PD patients\u003csup\u003e19\u003c/sup\u003e. Given this, we hypothesize that long-term activation of GCase in the brain may be a viable approach to attenuate the accumulation of \u0026alpha;-syn oligomers as induced by pathogenic LRRK2 mutation in PD. Ambroxol (ABX) (trans-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol; CAS number 18683-91-5) is a common medication for respiratory conditions, being utilized as a potential pharmacological chaperone for GCase\u003csup\u003e20\u003c/sup\u003e. Its therapeutic role in inducing GCase activity has been widely investigated, particularly in the context of Gaucher Disease (GD) and PD\u003csup\u003e20\u003c/sup\u003e. Oral administration of ABX is effective to penetrate through the blood-brain barrier (BBB) in nonhuman primates, resulted in increased GCase activity in key brain regions including cortex and striatum\u003csup\u003e21\u003c/sup\u003e. In our earlier studies, we revealed \u0026alpha;-syn oligomer accumulation in cortex and striatum of LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice starting at 14 months of age (equivalent to 38\u0026ndash;47 years old in human) and significantly increased by 18 months (equivalent to 56\u0026ndash;69 years old in human), compared to their age-matched WT littermates. We found that long-term therapeutic inhibition of mutant LRRK2 kinase hyperactivity in these mutant mice over 18 weeks significantly reduced brain \u0026alpha;-syn oligomer level\u003csup\u003e22\u003c/sup\u003e. Here, we aimed to use this model to test our hypothesis whether long-term spontaneous feeding of GCase activator (ABX) could attenuate accumulation of toxic \u0026alpha;-syn oligomers in aged LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mouse brain by inducing brain GCase activity and lysosomal degradation. Our findings further validated the importance of \u0026alpha;-syn oligomers as a promising modifiable therapeutic target in the development of PD therapies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTreatment of GCase activator (ABX) increased cellular GCase activity and reduced level of Ser129-\u0026alpha;-syn phosphorylation without affecting total\u0026nbsp;\u003c/strong\u003ea\u003cstrong\u003eSyn expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReduced GCase activity in PD is linked to \u0026alpha;-syn oligomerization and aggregation\u003csup\u003e19\u003c/sup\u003e. In particular, phosphorylation of \u0026alpha;-syn at residue Ser129 (pSer129) has been proposed to be a surrogate marker of synucleinopathies in PD\u003csup\u003e23\u003c/sup\u003e. We initially determined whether inducing cellular GCase activity by therapeutic GCase activator, ABX, could modulate Ser129-\u0026alpha;-syn phosphorylation in human neuronal cells. Normal SH-SY5Y cells express \u0026alpha;-syn protein at very low level. To better elucidate the ABX effect, cells were engineered to stably overexpress WT \u0026alpha;-syn by lentivirus transduction and selection. For cell treatment, cells at 70% confluence were refreshed with new medium for 2 hr before ABX treatment at 0, 10 and 50 \u0026micro;M for an additional 72 hr (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). Total cellular GBA levels and GCase activity were determined by Western blots and standard 4-MU fluorescence assay in the total cell lysates, respectively. Dose-dependent increase of GCase activity and GBA protein expression was observed in both normal and \u0026alpha;-syn overexpressing cells (all p\u0026lt;0.05; N=7), ranging from 10 to 50\u0026micro;M ABX treatment (all p\u0026lt;0.05; N=7) (\u003cstrong\u003eFig. 1b, c\u003c/strong\u003e). The cellular GCase activity in 50\u0026micro;M ABX treatment on both normal and \u0026alpha;-syn overexpressing cells were significantly increased by 24.2% and 16.9% compared to DMSO-treated controls (p\u0026lt;0.01; N=7) (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). Moreover, GBA protein levels in normal and \u0026alpha;-syn overexpressing cells were determined by western blot, showing significantly increased by 51.6% (p\u0026lt;0.01) and 33.4% (p\u0026lt;0.05) compared to DMSO-treated controls, respectively (\u003cstrong\u003eFig. 1c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFurthermore, western blot analysis showed that pSer129-\u0026alpha;-syn levels in cells treated with ABX at 50 \u0026micro;M were significantly reduced by 16.9% compared to DMSO-treated controls (p\u0026lt;0.01; N=7) (\u003cstrong\u003eFig. 1c\u003c/strong\u003e). Total \u0026alpha;-syn level was not affected by ABX treatment at all doses indicating that the reduction of pSer129-\u0026alpha;-syn levels were not due to changes in total cellular protein levels (\u003cstrong\u003eFig. 1c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eABX rescued GCase deficiency and suppressed Ser129-\u0026alpha;-syn phosphorylation in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mouse embryonic fibroblasts (MEFs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe property of ABX to reduce Ser129-\u0026alpha;-syn phosphorylation in human SH-SY5Y cells \u003cstrong\u003e(Fig. 1)\u0026nbsp;\u003c/strong\u003ehighlighted the therapeutic potential of modulating GCase activity to attenuate synucleinopathies in PD. We further validated if ABX similarly induced GCase activity in a parkinsonian LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant cell model. First, the basal cellular GCase activity was compared between WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs under normal culture condition. The gene mutation effect was evident by a significant 40.4% decrease in the GCase activity in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs compared to WT control (p\u0026lt;0.01; N=6), indicating GCase deficiency in the mutant. ABX treatment (50 \u0026mu;M) for 24 hr significantly attenuated such GCase deficiency in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs by 21.6% increase in activity compared to DMSO-treated mutant controls \u003cstrong\u003e(Fig. 2a)\u0026nbsp;\u003c/strong\u003e(p\u0026lt;0.05; N=6).\u003c/p\u003e\n\u003cp\u003eGiven that the GCase deficiency in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs was significantly attenuated by ABX, we further investigated the effect of ABX on suppressing pSer129 in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs which we engineered to stably overexpress mouse \u0026alpha;-syn\u003csup\u003e24\u003c/sup\u003e. These mutant MEFs were derived from our LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice showing age-dependent accumulation of \u0026alpha;-syn oligomers in their brains\u003csup\u003e24,25\u003c/sup\u003e. \u0026nbsp;To address ABX drug effects, \u0026alpha;-syn-overexpressing LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs were treated with ABX at 50\u0026micro;M for 24 hr before subjected to GCase activity assay and Western blotting (\u003cstrong\u003eFig. 2c\u003c/strong\u003e). GCase activity assay showed that ABX treatment significantly increased GCase activity in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs by 76.98% compared to DMSO-treated control cells (p\u0026lt;0.01, N=4) (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). Similar to the observation in SH-SY5Y cells, ABX treatment also significant reduced level of Ser129-\u0026alpha;-syn phosphorylation by 84.33% compared to the DMSO-treated control cells (p\u0026lt;0.01, N=4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eABX is thermally stable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rationale of testing the thermal stability of ABX was because we aimed to determine the therapeutic benefits of ABX in mice using long-term spontaneous feeding instead of multiple oral gavages to avoid unnecessary trauma. During the drug regimen preparation, pure ABX powder was mixed with standard rodent food pellets and re-molded which involved a drying step at 70\u0026deg;C for 30 min. in oven. Thus, we determined the thermal stability of ABX efficacy in activating GCase activity and suppression of Ser129-\u0026alpha;-syn\u0026nbsp;phosphorylation after heat shock in oven. The stock ABX solution dissolved in DMSO (50mM) was heated at\u0026nbsp;80\u0026deg;C for 30 min. before cooled down and subjected to MEF treatment. Similar to the ABX without heat shock, heated ABX retained similar activation of GCase activity (77.20%, p\u0026lt;0.01, N=4) and reduction of Ser129-\u0026alpha;-syn\u0026nbsp;phosphorylation\u0026nbsp;(70.56%, p\u0026lt;0.01, N=4), compared to DMSO-treated controls (\u003cstrong\u003eFig. 2b-e\u003c/strong\u003e), indicating that the efficacy of ABX is not affected by heat. The level of GCase activation by heat-treated ABX was comparable to the levels of ABX without heat treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpaired lysosomal degradation in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs was attenuated by ABX treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies showed that \u0026alpha;-syn aggregates are predominantly metabolized by lysosomes\u003csup\u003e26,27\u003c/sup\u003e, we further determined whether increasing GCase activity by ABX treatment modulated lysosomal protease activity. First, a real-time flow cytometry assay was developed to compare lysosomal protease activity in WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant cells using a self-quenched fluorescent substrate, DQ-BSA. Multiple clones of LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs were used to avoid confounding clonal effects. After ABX treatment, the intensity of emitted fluorescence upon DQ-BSA degradation in lysosomes was measured at different time points within 60 min to generate a time-dependent increase in fluorescence readouts (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). Our results showed that total lysosomal activity in mutant LRRK2 MEFs was significantly lower than that of WT controls by 57.9% as shown by AUC analysis (p\u0026lt;0.05; N=4) (\u003cstrong\u003eFig. 3b\u003c/strong\u003e). Treatment of ABX (50\u0026micro;M) significantly attenuated the impaired lysosomal activity by increasing lysosomal activity in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs by 30.4% (p\u0026lt;0.001; N=6). In contrast, similar ABX treatment in WT MEFs did not cause a significant effect on lysosomal activity (\u003cstrong\u003eFig. 3c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain penetration of ABX and drug clearance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaving our \u003cem\u003ein vitro\u003c/em\u003e findings showing ABX effects on GCase activity, we further investigated the drug benefits in our parkinsonian LRRK2 mutant mice. To avoid confounding effects of trauma from multiple oral gavages, we determined the therapeutic benefits of ABX in mice using long-term dose-controlled spontaneous feeding approach. Before long-term treatment, we determined whether oral ABX can achieve effective drug delivery to the mouse brain. First, we determined the maximum ABX concentration using LC-MS/MS achievable in serum, brain, and liver after a single oral gavage of ABX at 0.4 mg/g body weight (dissolved in physiological saline), which resulted in a peak concentration of ABX at 14.15\u0026plusmn;6.63\u0026micro;g/g, 199.78\u0026plusmn;117\u0026micro;g/g, and 123.05\u0026plusmn;35.96\u0026micro;g/g tissue in serum, brain, and liver, respectively, at 4 hr after oral gavage (p\u0026lt;0.05; N=3). The ABX level in brain, liver and serum subsequently declined to undetectable level at 48 hr post treatment (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). Next, we tested for the availability of ABX using spontaneous feeding for 14 days. Aged (14-month-old) WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice were fed \u003cem\u003ead libitum\u003c/em\u003e with ABX-containing food pellets (3.47mg ABX/g food powder) for 14 consecutive days. On the last day of ABX treatment, the mice were euthanized before brain, liver and serum were collected for ABX quantification by LC-MS/MS. Our results showed that ABX was detected in both brain and liver after 14-day ABX treatment protocol (\u003cstrong\u003eFig. 4b\u003c/strong\u003e). It is noteworthy that the average ABX concentration in the brain of LRRK2 mutant mice (28.08\u0026plusmn;5.9\u0026mu;g/g) was significantly higher than the level in WT mice (12.3\u0026plusmn;2.2\u0026mu;g/g) (p\u0026lt;0.05; N=6). However, no statistically significant difference was observed in both liver (WT: 16.86\u0026plusmn;2.39\u0026mu;g/g; R1441G: 21.89\u0026plusmn;7.19\u0026mu;g/g) and serum (WT: 1.26\u0026plusmn;0.46\u0026mu;g/g; R1441G: 1.47\u0026plusmn;0.38\u0026mu;g/g) between WT and LRRK2 mutant mice after the 14-day ABX treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong-term (18-week) spontaneous feeding of ABX reduced cumulative food consumption without significant changes in average body weight\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the long-term therapeutic effects of ABX, aged (14-month-old) WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (N\u0026ge;20 animals per treatment group) were fed with food pellets infused with known amount of ABX (3.47mg ABX/g food powder) for 18 weeks. Total food consumption of each mouse was recorded per week for estimation of weekly ABX intake per mouse. The body weight of individual mouse was recorded and plotted over the course of treatment. Both WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice maintained a consistent body weight over 18-week treatment (\u003cstrong\u003eFig. 5a\u003c/strong\u003e). Also, ABX treatment did not cause significant difference in body weight in both WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice compared to their respective controls without ABX. The weekly food consumption of ABX treatment in WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice showed no significant difference and maintained consistently over 18 weeks of spontaneous feeding (\u003cstrong\u003eFig. 5b\u003c/strong\u003e) Two-way ANOVA showed that LRRK2 mutation did not cause any significant effect on cumulative food consumption over 18-week of treatment. However, a significant effect of ABX treatment was observed on cumulative food consumption [F (1,32) = 20.56; p\u0026lt;0.001] as indicated by a lower cumulative food consumption in both WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (\u003cstrong\u003eFig. 5c\u003c/strong\u003e). Post-hoc comparisons showed that ABX treatment significantly reduced food consumption in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice (p\u0026lt;0.01; N=20), but not in WT mice (\u003cstrong\u003eFig. 5c\u003c/strong\u003e). Based on the amount of weekly food consumption in each mouse, the average weekly dose of ABX (mg/kg/week) in 18-week treatment was calculated, showing a similar level of average weekly dosage of 325.8\u0026plusmn;23.68 and 321.7\u0026plusmn;26.50 mg/kg/week in WT and LRRK2 mutant mice, respectively (N\u0026ge;20) over the whole course of treatment (\u003cstrong\u003eFig. 5d\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong-term spontaneous feeding of ABX increased GCase activity in striatum without affecting GBA protein levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter we confirmed the long-term feeding protocol, we investigated whether ABX treatment can induce GCase activity in striatum, the most susceptible brain region to synaptic dysfunction and neurodegeneration in early PD. The relationship of ABX and brain GBA protein level was investigated since GBA gene encoded for GCase. GCase activity assay showed that striatal GCase activity of LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice was significantly lower than that of WT controls by 13.1% (p\u0026lt;0.05; N\u0026sup3;16). However, the total cellular GBA level in striatum did not show significant difference between these two mouse lines (\u003cstrong\u003eFig. 6a\u003c/strong\u003e). More importantly, 18-week ABX treatment significantly increased GCase activity in WT by 21.6% (p\u0026lt;0.05; N\u0026ge;17) and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice by 40.6% (p\u0026lt;0.01; N\u0026ge;16) (\u003cstrong\u003eFig. 6a\u003c/strong\u003e). ABX treatment did not affect total GBA level in striatum of both WT and mutant mice (\u003cstrong\u003eFig. 6b\u003c/strong\u003e), indicating that ABX-induced GCase activation was not due to increased protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong-term spontaneous feeding of ABX did not affect Ser129\u003c/strong\u003e-\u003cstrong\u003e\u0026alpha;-syn\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ephosphorylation levels in striatum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhosphorylation of \u0026alpha;-syn at residue Ser129 (pSer129) has been considered a surrogate marker of synucleinopathies in PD\u003csup\u003e23\u003c/sup\u003e. To explore whether ABX-induced GCase activation modulated\u0026nbsp;\u0026alpha;-syn\u0026nbsp;phosphorylation, we assessed the levels of pSer129-\u0026alpha;-syn\u0026nbsp;in striatum\u0026nbsp;by western blotting in WT and LRRK2 mutant mice\u0026nbsp;after 18-week ABX feeding. Western blot analysis showed that the basal level of pSer129 in striatum were similar in WT and LRRK2 mutant mice (N\u0026ge;16) (\u003cstrong\u003eFig. 7a\u003c/strong\u003e). The total\u0026nbsp;\u0026alpha;-syn level as determined by ELISA also showed no statistically significant difference between the two mouse lines\u0026nbsp;(N\u0026ge;16).\u0026nbsp;Spontaneous feeding of ABX over 18 weeks also did not cause any significant effects on pSer129 level in striatum of both WT (N\u0026ge;17) and mutant mice (N\u0026ge;16) (\u003cstrong\u003eFig. 7b\u003c/strong\u003e). This \u003cem\u003ein vivo\u003c/em\u003e observation contrasted the effects of ABX which significantly suppressed Ser129 phosphorylation seen in SH-SY5Y and MEFs (\u003cstrong\u003eFig. 1 \u0026amp; Fig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong term spontaneous feeding of ABX reduced soluble\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026alpha;-syn\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;oligomer levels in mutant LRRK2\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;R1441G\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mouse striatum\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePathogenic LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutation has been associated with increased accumulation of \u0026alpha;-syn oligomers in the brain\u003csup\u003e24\u003c/sup\u003e.\u0026nbsp;Although ABX treatment did not show a significant effect on striatal Ser129-\u0026alpha;-syn\u0026nbsp;phosphorylation, we further investigated the therapeutic effects on\u0026nbsp;\u0026alpha;-syn\u0026nbsp;oligomer level in the brain.\u0026nbsp;\u0026alpha;-syn oligomers in cortex and striatum of ABX treated mice were quantified using a validated, conformational-specific ELISA. Briefly, brain tissues were freshly lysed by sonication in detergent-free PBS supplemented with protease inhibitors to extract the total cell lysates containing soluble \u0026alpha;-syn oligomers. Total oligomer levels (pg/mg total protein) in target brain regions were quantified based on linear standard curve provided by the kit, and normalized by mg total cellular protein. Our ELISA results showed that total soluble \u0026alpha;-syn oligomers levels in both striatum and cortex were significantly higher in untreated LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice compared WT control mice (79.7%, 20.6%; p\u0026lt;0.01; N\u0026ge;16) (\u003cstrong\u003eFig. 8\u003c/strong\u003e), consistent with our earlier findings\u003csup\u003e24\u003c/sup\u003e. Following 18-week spontaneous feeding of ABX, striatal\u0026nbsp;\u0026alpha;-syn\u0026nbsp;oligomer levels in ABX-treated mutant mice were significantly\u0026nbsp;reduced (23.4%; p\u0026lt;0.05; N\u0026ge;16), indicating that our long-term treatment regimen was efficacious in reducing abnormal accumulation of\u0026nbsp;\u0026alpha;-syn\u0026nbsp;oligomer levels in mutant to normal physiological levels comparable to those in WT mice (\u003cstrong\u003eFig. 8a\u003c/strong\u003e). Unlike the effects in the striatum, long-term ABX treatment did not reduce cortical oligomer levels in mutant LRRK2 mice (\u003cstrong\u003eFig. 8b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe correlation between GCase dysfunction and PD pathogenesis is increasingly recognized as a critical factor underlying nigrostriatal dopaminergic neurodegeneration. Mutations in the GBA1 gene, which encodes GCase, constitute the most significant known genetic risk factor for the development of PD. Clinical studies showed that GCase expression and activity are both decreased in idiopathic PD brain regions (e.g. caudate and substantia nigra) where they accumulate misfolded \u0026alpha;-syn protein\u003csup\u003e19\u003c/sup\u003e. This relationship is underscored by findings showing that GCase deficiency enhances \u0026alpha;-syn aggregation in PD\u003csup\u003e28\u003c/sup\u003e. Our current findings consolidated this pathological relationship between GCase activity and synucleinopathies in the context of PD, particularly on the role of soluble oligomeric forms of \u0026alpha;-syn. In our study, we observed that the lysosomal function and GCase activity were reduced in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant MEFs and mice striatum compared to their WT littermates, reflecting similar clinical situations in human PD. Our results demonstrated that GCase activity and lysosomal function were significantly enhanced following ABX administration in both \u003cem\u003ein vitro\u003c/em\u003e cell models (human SH-SY5Y and MEFs) and in aged LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice. Previously, we elucidated an age-dependent accumulation of \u0026alpha;-syn oligomers in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mouse striatum, which mirrors key prodromal phenotypic changes of PD. This age-dependent phenomenon began in mutant mice at 14 months of age, with oligomer levels rising progressively by 18 months\u003csup\u003e24\u003c/sup\u003e. To test our hypothesis whether activation of GCase can modulate \u0026alpha;-syn oligomer level in the brain, an 18-week spontaneous feeding regimen of ABX was given to 14-month-old LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice to induce GCase activity and lysosomal function. Our treatment protocol achieved an average weekly dose at 300mg/kg of ABX based on individual mouse weekly food consumption. This dosage level is much lower than the reference LD\u003csub\u003e50\u003c/sub\u003e of ABX administered orally in mice at 2380 mg/kg (\u003cem\u003eRegistry of Toxic Effects of Chemical Substances (RTECS), The Dictionary of Substances and their Effects, 1st Edition, IUCLID\u003c/em\u003e). Our current dose showed significant increase of GCase activity in the mutant mouse brains, where the \u0026alpha;-syn oligomer level was concomitantly reduced. Given that higher-ordered \u0026alpha;-syn aggregates are predominantly metabolized via autophagic lysosomal degradation\u003csup\u003e29\u003c/sup\u003e, this is the first study to demonstrate that long-term therapeutic activation of GCase and lysosomal activity can attenuate accumulation of toxic \u0026alpha;-syn oligomers in aged brains. Our findings also reiterate the role of soluble \u0026alpha;-syn oligomers as a modifiable therapeutic target to attenuate synucleinopathies in PD.\u003c/p\u003e\n\u003cp\u003eAs a proof concept, we utilized ABX as a known therapeutic GCase activator for cell and mouse treatment. ABX is a known drug that can break up phlegm in the treatment of respiratory diseases associated with viscid or excessive mucus, e.g., bronchitis or asthma. Here, we tested whether long-term feeding of ABX could maintain an elevated level of brain GCase activity and showed beneficial effects to reduce \u0026alpha;-syn oligomer accumulation in our aged LRRK2 mutant mouse brains. We first considered the route of drug administration by spontaneous feeding which would not cause trauma or stress to the testing animals over a long period of treatment time. We prepared the mouse feed with a known amount of ABX based on an estimation of 7 grams of daily food pellet consumption per mouse (C57BL/6 mouse). After 18-week of spontaneous feeding, the average body weight of the treated mice showed no significant difference compared to untreated mice. This indicated that our feeding protocol is feasible to control ABX dosage and avoid unnecessary trauma caused otherwise by multiple daily oral gavages over 18-week. It is noteworthy that each mouse consumed a slightly different amount of food per day. However, since ABX was fed over a long period of time, the average weekly dose of ABX in both WT and LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice were comparable without significant differences. This average weekly dose level of ABX achieved significantly increased GCase activity in the brain after 18-week treatment regimen.\u003c/p\u003e\n\u003cp\u003eAlthough the molecular link between GCase activity and \u0026alpha;-syn accumulation in PD brain is still unclear, our data supported our hypothesis that the observed reduction in \u0026alpha;-syn oligomer levels in ABX-treated mouse brains can be attributed to the increased therapeutic activation of GCase, which may subsequently facilitate degradation of \u0026alpha;-syn aggregates through improved lysosomal function. Previous studies linked GCase dysfunction with \u0026alpha;-syn accumulation by showing that viral-mediated suppression of GCase activity in Gaucher\u0026rsquo;s disease mice led to an increase in \u0026alpha;-syn pathology\u003csup\u003e30,31\u003c/sup\u003e. Conversely, activation of GCase has been shown to improve the clearance of pathological \u0026alpha;-syn, which supports the idea of therapy targeting the accumulation of toxic species attributed to PD pathology\u003csup\u003e32\u003c/sup\u003e. These outcomes underscore the validity of GCase as a therapeutic target, particularly in the context of genetic variants such as LRRK2 mutations, which are associated with predisposed dysregulation of \u0026alpha;-syn pathways. Hyper-phosphorylated \u0026alpha;-syn is one of the major components found in the Lewy body of PD brains\u003csup\u003e33\u003c/sup\u003e. Phosphorylated Ser129-\u0026alpha;-syn is a proposed surrogate marker for PD diagnosis. Abnormal phosphorylation of the protein has been shown to increase the propensity of its oligomer formation \u003cem\u003ein vitro\u003c/em\u003e\u003cem\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eHowever, the level of pSer129-\u0026alpha;-syn in striatum of our aged LRRK2 mutant mice did not exhibit significant changes after 18-week ABX treatment, yet the oligomer \u0026alpha;-syn level was reduced by a significant 23%. This finding suggests that while GCase activation effectively reduced the accumulation of \u0026alpha;-syn oligomers, it did not significantly impact the phosphorylation status of Ser129-\u0026alpha;-syn in the brain. The reduction in oligomer levels without a change in phosphorylation suggests that the GCase activation may promote the clearance or disaggregation of \u0026alpha;-syn oligomers rather than inhibited oligomer formation. This argument is supported by the significant increase of lysosomal activity in LRRK2 mutant MEFs following ABX treatment. Moreover, our previous studies elucidated lysosomal dysfunction in LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice. It is plausible that therapeutic activation of GCase enhanced lysosomal activity that facilitated more efficient degradation of \u0026alpha;-syn oligomers via activating the lysosomal autophagic pathways in these mutant animals. Furthermore, since glucosylceramide (GlcCer) accumulation is known to promote \u0026alpha;-syn aggregation by stabilizing \u0026alpha;-syn oligomers\u003csup\u003e35\u003c/sup\u003e, increasing GCase activity could reduce GlcCer level that subsequently destabilized existing aggregates for downstream lysosomal degradation. The lack of change in pSer129-\u0026alpha;-syn levels suggests the ABX intervention preferentially targeted oligomer clearance rather than modulating phosphorylation-dependent aggregation of \u0026alpha;-syn.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImproving GCase function while controlling \u0026alpha;-syn oligomer levels appears to be a sensible approach to delay neurodegenerative processes linked with PD. Recent studies focused on improving GCase activity as the therapeutic strategies by using the GCase activator compounds including ABX. Supportive to our findings, these compounds improved GCase activity in neurons derived from PD patients with GBA mutations\u003csup\u003e30\u003c/sup\u003e. Besides ABX, a study applied small molecule S-181 as the GCase modulator on iPSC-derived dopaminergic neurons from PD patients with GBA1 mutation and GBA1 mutant mice to enhance GCase activity. The results showed that the GCase activity was increased while the accumulation of \u0026alpha;-syn was reduced in these patient neurons \u003cem\u003ein vitro\u003c/em\u003e\u003cem\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/em\u003e. The current landscape of clinical research underscores the potential of GCase activators as disease-modifying therapies for PD by improving lysosomal function and mitigating neurodegenerative changes induced by \u0026alpha;-syn accumulation. Currently, clinical studies are underway using GCase activator for the treatment of PD, mainly in patients carrying GBA1 mutation. For instance, Vanqua Bio announced positive interim results from phase 1 clinical trial of VQ-101, an orally administered, brain-penetrant, allosteric activator of GCase for the treatment of GBA-PD\u003csup\u003e37\u003c/sup\u003e, which aimed to reduce \u0026alpha;-syn accumulation. These studies highlight the growing interest in GCase activators to target the underlying lysosomal dysfunction in PD. Our current findings highlight the benefits of chronic therapeutic GCase activation to reduce progressive accumulation of \u0026alpha;-syn oligomers in LRRK2-associated PD. Such pathology was confidently shown in PD patients by two recent independent clinical studies\u003csup\u003e17,18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study demonstrated for the first time a feasible therapeutic approach to induce GCase activity, showing a concomitant reduction of \u0026alpha;-syn oligomer accumulation in the brains. Our aged LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mutant mice are well-characterized disease models demonstrating progressive accumulation of toxic soluble \u0026alpha;-syn oligomers in the brains with age. This model is valuable to reveal molecular events at the prodromal stage of PD for testing early therapeutic options. The therapeutic outcomes from long-term spontaneous feeding of GCase activator in our mutant mice shed light on new strategies to attenuate synucleinopathies by inducing GCase activity and lysosomal degradation of toxic oligomers in PD brains. Although ABX has well-documented drug safety profile in human as medicine, further investigation using other brain-penetrant GCase activators is needed to confirm the specific effect of GCase activation and its modulation on \u0026alpha;-syn pathologies. Future investigations include elucidation of the mechanism(s) regarding the combined effects of GCase activation and \u0026alpha;-syn post-translational modifications. By addressing these disease-related pathways, we may enhance the capacity to alter disease progression related to synucleinopathies and potentially improve clinical outcomes for PD. Our recent report on therapeutic LRRK2 inhibition is a vivid example to attenuate aberrant LRRK2 kinase hyperactivity that modulated cellular clearance of \u0026alpha;-syn oligomers\u003csup\u003e22\u003c/sup\u003e. This could be a novel approach by combining GCase activator and LRRK2 inhibitor that ameliorates aberrant LRRK2 kinase hyperactivity and reduces the accumulation of \u0026alpha;-syn oligomers, the emerging toxic species that were previously underestimated in PD pathogenesis and treatment.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLRRK2\u003csup\u003eR1441G\u003c/sup\u003e homozygous knockin mouse model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA C57BL/6 mice with complete homozygous knockin of pathogenic LRRK2\u003csup\u003eR1441G\u0026nbsp;\u003c/sup\u003emutation (cDNA 4321C\u0026gt;G) maintained under pure C57BL/6N mouse background\u003csup\u003e38\u003c/sup\u003e. All mice were housed in the Laboratory Animal Unit, HKU with accreditation through the Association for Assessment and Accreditation of Laboratory Animal care international (AAALAC), under standard conditions (12-h light/dark cycle) with unrestricted access to food and water. The procedure of experimental use of animals was approved by the Institutional Animal Care and Use Committee, HKU (CULATR#4506-17).\u0026nbsp;All\u0026nbsp;studies involved followed the ARRIVE guidelines. Genotype of animals was determined by DNA sequencing. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMouse embryonic fibroblasts (MEFs) culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHomozygous LRRK2\u003csup\u003eR1441G\u003c/sup\u003e knockin mutant mice carrying mutation (cDNA 4321 C\u0026gt;G) of LRRK2 and their wild-type littermates were used for preparation of mouse embryonic fibroblasts (MEFs) according to our published protocol\u003csup\u003e24\u003c/sup\u003e. Individual mouse embryos at day E12.5 were isolated from crosses between two heterozygous LRRK2\u003csup\u003eWT/R1441G\u003c/sup\u003e mice\u003csup\u003e24\u003c/sup\u003e. Individual clone of MEFs was obtained from each embryo and genotyped at passage 1 when developed into proliferating culture. All MEFs were cultured in Dulbecco\u0026rsquo;s Modified Eagle medium (DMEM; ThermoFisher\u0026trade; Scientific, 10,569\u0026ndash;010) containing 15% Fetal Bovine Serum (FBS; GE Healthcare HyClone\u0026trade;, SH30071.03), 100 units/ml penicillin, 100 \u0026mu;g/mL streptomycin (ThermoFisher\u0026trade; Scientific, 15,140\u0026ndash;122), non-essential amino acids ThermoFisher\u0026trade; Scientific, 11,140\u0026ndash;050).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStable overexpression of human \u0026alpha;-syn in SH-SY5Y cell line\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effect of GCase activation on \u0026alpha;-syn-Ser129 phosphorylation, human \u0026alpha;-syn protein was stably overexpressed in SH-SY5Y cells (ATCC; CRL-2266) by transduction of lentivirus following our previous published protocol\u003csup\u003e22\u003c/sup\u003e. \u0026nbsp;All SH-SY5Y cell lines were cultured in Dulbecco\u0026apos;s Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, ThermoFisherTM Scientific, 11320-082), supplemented with 10% fetal bovine serum (FBS; GE Healthcare HyCloneTM, SH30071.03), 1% 100 units/ml penicillin, and 100 \u0026mu;g/mL streptomycin (Gibco, ThermoFisherTM Scientific, 15140\u0026ndash;122). \u0026nbsp;To generate \u0026alpha;-syn expression construct, human SNCA (NCBI Entrez Gene: 6622) gene was amplified by PCR from the total cDNA of SH-SY5Y cells as a template to generate an insert of cDNA fragment encoding for \u003cem\u003eh\u003c/em\u003eSNCA. This \u003cem\u003eh\u003c/em\u003eSNCA cDNA insert was then sub-cloned into a lentiviral backbone plasmid pSIN4-EF2-IRES-Pur derived from a gift (Addgene\u0026trade; plasmid, 16580) to construct pSIN4-\u003cem\u003eh\u003c/em\u003eSNCA plasmid (Addgene\u0026trade; plasmid, 102366). Expression levels of total and phosphorylated Ser129-\u0026alpha;-syn (pS129) were determined by Western blotting. Cells at 80% confluence were treated with a GCase activator, Ambroxol\u0026trade; (ABX;\u0026nbsp;A9797; Sigma Aldrich),\u0026nbsp;at 10 and 50\u0026micro;M for 72 hr before GCase activity assay and Western blotting. Negative control cells were treated with DMSO (v/v 0.01%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFeeding of ABX by food pellets in mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve long-term feeding of ABX without traumatizing the mice, ABX was mixed into food pellets for \u003cem\u003ead libitum\u003c/em\u003e feeding. Normal mouse diet (150g; PicoLab\u0026reg; Rodent Diet 20, LabDiet\u0026trade;, Cat. no 5053) was grinded into powder and then mixed with 520mg of ABX aiming to achieve the dose of 800mg/kg per day [the highest reported dose without noted toxic effect\u003csup\u003e39,40\u003c/sup\u003e] based on estimated daily consumption of 7 grams of food pellet per C57BL/6 mouse. 150ml of sterilized distilled water was added into the mixture of ABX and ground food powder and moulded into size similar to normal food pellet. The ABX-containing food pellets were dried at 70\u0026deg;C in oven overnight and stored at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTreatment of ABX in cell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo induce GCase activity, ABX was dissolved in dimethyl sulfoxide (DMSO) to produce a 50mM stock solution. ABX treatments and vehicle (DMSO) controls were done to achieve a 0.01% DMSO concentration (v/v) in culture medium. Unless otherwise specified, dissolved ABX remained in the culture medium until time of harvest without medium replacement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell Harvest and Immunoblotting\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreated cells (both SH-SY5Y and MEFs) were harvested through washing with culture medium and collection of a cell pellet through centrifugation at 4\u0026deg;C for 10 min at 2500 rpm. The medium was aspirated, and cell pellet was resuspended in 1x RIPA buffer without SDS (Cell Signaling Technology, 9803S) supplemented with Halt\u0026trade; Protease and Phosphatase Inhibitor Cocktail, 100x (ThermoFisher Scientific, 78446) and 2% phenylmethylsulfonyl fluoride (PMSF) (Pierce, 36978). Protein lysates were incubated on ice for 30 min, followed by centrifugation at 4\u0026deg;C for 15 min at 14000 rpm. Protein concentration was determined through Bradford Assay (Quick Start\u0026trade; Bradford 1x Dye Reagent, #5000205). Equal amounts of proteins were dissolved in sample buffer (Thermo Scientific\u0026trade; Pierce\u0026trade; Lane Marker Non-Reducing Sample Buffer, 39001), and electrophorized in 4% stacking, 12% resolving SDS-polyacrylamide gels, at 80V. The resulting gels were electro-transferred to PVDF membranes. The resulting membranes incubated in 4% paraformaldehyde solution in PBS (Santa Cruz Biotechnology, 30525-89-4) for 20 min, and was subsequently blocked in 5% bovine serum albumin (Sigma Aldrich, A9647) in TBST (Santa Cruz Biotechnology \u0026ndash; sc362311), and probed with primary antibodies overnight and HRP-conjugated secondary antibodies (P0260, Polyclonal rabbit anti-mouse immunoglobulins/HRP, Agilent DAKO\u0026trade;; or P0448, Goat Anti-Rabbit Immunoglobulins/HRP Agilent DAKO\u0026trade;) for one hour in 1% bovine serum albumin in TBST. This was followed by incubation in ECL substrate solution for chemiluminescence visualization. The quantification of band intensity was done using the Image Lab Software (BioRad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLysosomal activity assay\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA real-time flow cytometry assay was developed to compare lysosomal protease activity in WT and LRRK2 mutant cells. Multiple clones of LRRK2 mutant MEFs were used to avoid confounding clonal effects.\u0026nbsp;Cells in refreshed medium for 2 hr were treated with a lysosome protease substrate, DQ-BSA (10 \u0026micro;g/ml), for different time points. Intact DQ-BSA protein does not emit fluorescence\u003csup\u003e41\u003c/sup\u003e. However, once this protein substrate is degraded by protease in lysosomes, it is broken down into protein fragments with isolated fluorophores (red). After such de-quenching by DQ-BSA degradation in lysosomes, the fluorescent intensity in cells was measured by flow cytometry (excitation: 590 nm; emission: 620 nm). The increased amount of fluorescent DQ-BSA metabolites as calculated by the area-under-curve (AUC) analysis reflected the level of lysosomal activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGCase enzymatic activity assay for mouse brain tissues and cell lysates\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse brain and peripheral organ tissue samples were homogenized in cold PBS buffer by sonication with 1X protease inhibitor cocktail (ThermoFisher Scientific, 186284), 1X EDTA (ThermoFisher Scientific, 1861283) and 1mM phenylmethylsulfonyl fluoride (PMSF) (Pierce, 36978). Cell lysates were homogenized in 1X RIPA buffer without SDS (Cell Signaling Technology, 9803S) supplemented with 1X protease inhibitor cocktail and 2X PMSF. Homogenates were centrifuged at 4\u003csup\u003eo\u003c/sup\u003eC for 10 min at 14000rpm and protein concentration of the supernatant was measured using Bradford Assay (BioRad, Quick Start\u0026trade; Bradford Protein Assay, 5000205). Resulting lysate was diluted to 1\u0026micro;g/\u0026micro;l in McIlvaine buffer (pH 5.4, mixing of 0.1M citric acid (Sigma-Aldrich, 251275) and 0.2M disodium phosphate (Sigma-Aldrich, S-0876). GCase activity was measured by incubating 20\u0026micro;l of diluted lysate with 5mM 4-methylumbelliferyl-beta-d-glucopyranoside (Sigma-Aldrich, M3633) in 40\u0026micro;l McIlvaine buffer supplemented with 22mM sodium taurocholate hydrate (Sigma-Aldrich, 86339) at room temperature for 3 h (brain or peripheral organ samples) or for 30 min (cell lysates)\u003csup\u003e42\u003c/sup\u003e.Standard curve was generated from 250 \u0026micro;M to 1.95 \u0026micro;M of 4-methylumbelliferone (4-MU) (Sigma-Aldrich, M1381) dissolved in DMSO. The reaction was terminated by adding 50 \u0026micro;l of 1 M glycine buffer (Affymetrix, Glycine, 16407 5KG) (pH 10.0) which is dissolved in water, and the fluorescence from cleaved product (4-MU) was measured using a spectrophotometer (Ex\u003csub\u003e365\u003c/sub\u003e/Em\u003csub\u003e450\u003c/sub\u003e). All the samples were done in duplicate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGBA protein expression level in mouse brain tissues\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal glucosylceramidase (GBA) level in mouse brain was quantified using a commercial ELISA kit according to manufacturer\u0026rsquo;s protocol (MyBiosource.com, MBS7206378). Protein concentration of brain lysates was determined using Bradford assay. Standards were provided from the kit and diluted samples were added into anti-GBA antibody coated wells. The optical density (OD) at 450nm was measured by a microplate reader. The concentration of GBA was calculated by a four-parameter logistic (4-PL) curve-fit or logit-log linear regression curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTissue extraction and quantification of ABX using LC-MS/MS\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the bioavailability of ABX, we quantified the drug in mouse serum, liver and brain using LC-MS-MS. Briefly, ABX in serum, liver and brain samples was extracted by sonicating the homogenized samples for 20, 40 and 40\u0026thinsp;min with 4\u0026thinsp;ml of acetonitrile, respectively. The samples were then centrifuged at 10,000\u0026thinsp;rpm for 10\u0026thinsp;min to remove insoluble tissue debris. The supernatant was deproteinized and delipidated twice by the addition of 2\u0026thinsp;ml of acetonitrile and hexane, respectively. The clean acetonitrile layer was obtained after centrifugation at 10,000\u0026thinsp;rpm for 10\u0026thinsp;min. The clean acetonitrile layer was then collected for future analysis. An Agilent (Palo Alto, CA) 1290 Infinity liquid chromatograph coupled to a SCIEX QTRAP 3200 tandem mass spectrometer (Woodlands, Singapore) with an electrospray ionization interface was used. The analytical column is ZORBAX Eclipse Plus C18 (2.1\u0026thinsp;\u0026times;\u0026thinsp;100\u0026thinsp;mm, 1.8\u0026thinsp;\u0026micro;m, Agilent) equipped with its corresponding guard column (5\u0026thinsp;\u0026times;\u0026thinsp;2.1\u0026thinsp;mm, 1.8\u0026thinsp;\u0026micro;m, Agilent). The elution was conducted under isocratic conditions with a mobile phase composed of 90% methanol (with 10\u0026thinsp;mM ammonium acetate) and 10% Milli-Q water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantification of total and oligomeric \u0026alpha;-syn level\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe oligomeric \u0026alpha;-syn level in the mouse brain lysates after 18-week ABX \u003cem\u003ead libitum\u003c/em\u003e feeding were quantified using a commercial ELISA kit with assay sensitivity of 1.0pg/ml (MBS724099; MyBioSource.com\u0026trade;) according to our published protocol\u003csup\u003e22\u003c/sup\u003e. Mouse brain tissues were freshly homogenized in ice-cooled PBS supplemented with Halt\u0026trade; Protease and Phosphatase Inhibitor (ThermoFisher\u0026trade; Scientific, 78444), PMSF (Pierce\u0026trade;, #36978) and EDTA to preserve the native \u0026alpha;-syn oligomer. Homogenates were briefly centrifuged for 5 min at 720 \u0026times; g to remove fatty tissues and nuclei. Resultant supernatants containing cytoplasmic fraction and small cellular organelles were further clarified by centrifugation at 4 \u0026deg;C for 15 min at 12,000 \u0026times; g. Cytosolic fractions containing soluble \u0026alpha;-syn oligomers were collected from the resultant supernatants. Protein concentrations of PBS-soluble lysates were determined by Bradford assay (ThermoFisher\u0026trade; Scientific, #5000205). Briefly, 500\u0026ndash;700 \u0026mu;g of total lysates were subjected to \u0026alpha;-syn oligomer ELISA. Quantification of \u0026alpha;-syn oligomer levels in PBS-soluble lysates (pg oligomers/mg total protein) was based on the linear standard curve generated from recombinant \u0026alpha;-syn oligomer standards provided by the kit. Total \u0026alpha;-syn was quantified by another commercial ELISA kit (ThermoFisher Scientific\u0026trade;; KHB0061) according to manufacturer\u0026rsquo;s protocol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed based on independent trials to achieve statistical significance, as indicated in figure legends. Results were expressed as means \u0026plusmn; SEM. Conclusions were drawn based on statistical analyses using GraphPad\u0026trade; PRISM software (GraphPad Inc., CA). The normality of data sets was determined using D\u0026rsquo;Agostino \u0026amp; Pearson omnibus normality test. Potential outliers were identified using Grubb\u0026rsquo;s test. LRRK2 mutation effect and differences between the ABX-treatment and untreated control groups were determined by unpaired Student\u0026rsquo;s t-test to demonstrate drug effect. Alternative analysis was performed using non-parametric, Mann\u0026ndash;Whitney U-test to compare differences between two independent groups when the dependent variables did not fulfill data normality assumptions. Group comparisons were considered significant when p-value was less than 0.05 (p \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no data in this paper that can be submitted to a data repository. The data that support the findings in this article are available from the corresponding author on request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Tai Hung Fai Charitable Foundation - Edwin S H Leong Research Programme for Parkinson\u0026rsquo;s Disease (PWL Ho; #P0054772) for their long-term trust and funding support. We also acknowledge funding support by Health and Medical Research Fund (HMRF; #07183516), Health Bureau, Hong Kong SAR, China. Postdoctoral Fellowship (E.E.S. Chang) was supported by SKLMP Seed Collaborative Research Fund, State Key Laboratory of Marine Pollution, City University of Hong Kong (CityU), and Postdoc Matching Fund Scheme 23/24 (P0050969), The Hong Kong Polytechnic University (HK PolyU; Fund holder: P.W.L.H.). Research consumables and staff cost were partly supported by Start-up Fund for New Recruit, HK PolyU (Fund holder: P.W.L.H.), and an internal department fund by Department of Rehabilitation Sciences, HK PolyU (Fund holder: Benjamin K. Yee, Co-I: P.W.L.H.). Our appreciation of technical assistance by Dr.\u0026nbsp;Phoebe Ruan, and Qi Wang\u003csup\u003e\u0026nbsp;\u003c/sup\u003efrom CityU on LC-MS/MS measurements and data analysis. The authors also thank for the administrative supports from the Department of Rehabilitation Sciences, HK PolyU, and the Department of Medicine, The University of Hong Kong.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.W.L.H., Z.Y.K.C., H.L., and S.L.H. designed the project. Z.Y.K.C., H.L., E.E.S.C. P.R., Q.W., I.L.L., Y.M. and S.X.Y.Z performed experiments and data analysis. Z.Y.K.C. H.F.L. and B.W.M.L. managed laboratory logistics and safety. P.W.L.H. and S.Y.Y.P. oversaw and evaluated all experimental data. Z.Y.K.C., P.W.L.H. and S.Y.Y.P. \u0026nbsp;wrote and review the manuscript with input of all co-authors. All authors read and approved the final manuscript. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests, and all authors have agreed to publish this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah R.\u003cem\u003e, et al.\u003c/em\u003e Parkinson\u0026apos;s disease and age: The obvious but largely unexplored link. \u003cem\u003eExp Gerontol.\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 33-8 (2015). \u003c/li\u003e\n\u003cli\u003eKlein C. and Westenberger A. Genetics of Parkinson\u0026apos;s Disease. \u003cem\u003eCold Spring Harbor perspectives in medicine.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, a008888-a008888 (2012). \u003c/li\u003e\n\u003cli\u003eStoker T. 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Accumulation of toxic soluble a-syn seeds in patient cerebrospinal fluid represents a prodromal marker of synucleinopathies in PD, contributing to progressive neurodegeneration. Deficiency in beta-glucocerebrosidase (GCase), a lysosomal enzyme for glucocerebroside metabolism, is evident in PD linking functionally with pathogenic LRRK2 (leucine-rich repeat kinase 2) mutation and synucleinopathies. However, whether GCase activation ameliorates synucleinopathies in PD brains is unclear. Here, we explored how GCase activity affected Ser129-a-syn phosphorylation, and whether long-term treatment of a brain-penetrant GCase chaperone (Ambroxol; ABX) attenuated a-syn oligomer accumulation in aged mutant LRRK2\u003csup\u003eR1441G\u003c/sup\u003e mouse brains. Acute ABX treatment (50µM) significantly increased cellular GCase activity and reduced Ser129-a-syn phosphorylation in human SH-SY5Y neuronal cells and murine fibroblasts. Real-time DQ-BSA degradation assay revealed lysosomal dysfunction in mutant LRRK2\u003csup\u003eR1441G\u003c/sup\u003e MEFs, which was attenuated by ABX treatment. Single oral gavage of ABX (400mg/kg) in mice achieved peak drug level in serum and brain within 6 hours post-administration. Spontaneous feeding of ABX in food pellet over 18 weeks (average daily dose: 45.9mg/kg/day) elevated brain GCase activity in aged wildtype and mutant striatum without affecting body weight. This chronic regimen significantly reduced a-syn oligomer level in mutant striatum yet without an effect on total a-syn and Ser129-phosphorylation levels. This is the first study demonstrating attenuation of synucleinopathies by chronic GCase activation in aged mouse brains vulnerable to PD, suggesting early intervention to alter progression of synucleinopathies as a key determinant of clinical outcomes of PD.\u003c/p\u003e","manuscriptTitle":"Long-term oral regimen of glucocerebrosidase activator reduces a-synuclein oligomer accumulation in aged LRRK2 mutant mouse brains - therapeutic implication of Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 09:25:35","doi":"10.21203/rs.3.rs-7185990/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-30T11:16:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T15:51:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T07:58:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133722770112233047360636079872461903109","date":"2025-07-26T05:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172828213261674018556785137433393540584","date":"2025-07-26T02:33:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-25T02:34:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-23T20:10:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-23T12:49:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2025-07-22T10:34:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e75166f9-7ce6-4ac5-bc81-71ca5710fd7f","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52185394,"name":"Health sciences/Diseases"},{"id":52185395,"name":"Health sciences/Neurology"},{"id":52185396,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-12-15T16:15:19+00:00","versionOfRecord":{"articleIdentity":"rs-7185990","link":"https://doi.org/10.1038/s41531-025-01205-7","journal":{"identity":"npj-parkinsons-disease","isVorOnly":false,"title":"npj Parkinson's Disease"},"publishedOn":"2025-12-12 15:59:13","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-07-29 09:25:35","video":"","vorDoi":"10.1038/s41531-025-01205-7","vorDoiUrl":"https://doi.org/10.1038/s41531-025-01205-7","workflowStages":[]},"version":"v1","identity":"rs-7185990","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7185990","identity":"rs-7185990","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}