GBA1-Associated Parkinson's Disease Is a Distinct Entity.

GBA1-associated Parkinson's disease (GBA1-PD) is increasingly recognized as a distinct entity within the spectrum of parkinsonian disorders. This review explores the unique pathophysiological features, clinical progression, and genetic underpinnings that differentiate GBA1-PD from idiopathic Parkinson's disease (iPD). GBA1-PD typically presents with earlier onset and more rapid progression, with a poor response to standard PD medications. It is marked by pronounced cognitive impairment and a higher burden of non-motor symptoms compared to iPD. Additionally, patients with GBA1-PD often exhibit a broader distribution of Lewy bodies within the brain, accentuating neurodegenerative processes. The pathogenesis of GBA1-PD is closely associated with mutations in the GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase). In this review, we discuss two mechanisms by which GBA1 mutations contribute to disease development: 'haploinsufficiency,' where a single functional gene copy fails to produce a sufficient amount of GCase, and 'gain of function,' where the mutated GCase acquires harmful properties that directly impact cellular mechanisms for alpha-synuclein degradation, leading to alpha-synuclein aggregation and neuronal cell damage. Continued research is advancing our understanding of how these mechanisms contribute to the development and progression of GBA1-PD, with the 'gain of function' mechanism appearing to be the most plausible. This review also explores the implications of GBA1 mutations for therapeutic strategies, highlighting the need for early diagnosis and targeted interventions. Currently, small molecular chaperones have shown the most promising clinical results compared to other agents. This synthesis of clinical, pathological, and molecular aspects underscores the assertion that GBA1-PD is a distinct clinical and pathobiological PD phenotype, necessitating specific management and research approaches to better understand and treat this debilitating condition.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Fragment-Based Discovery of a Series of Allosteric-Binding Site Modulators of ß-Glucocerebrosidase.

ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions.

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency.

Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Peripheral Upregulation of Parkinson's Disease-Associated Genes Encoding α-Synuclein, ß-Glucocerebrosidase, and Ceramide Glucosyltransferase in Major Depression.

Due to the high comorbidity of Parkinson's disease (PD) with major depressive disorder (MDD) and the involvement of sphingolipids in both conditions, we investigated the peripheral expression levels of three primarily PD-associated genes: α-synuclein (SNCA), lysosomal enzyme ß-glucocerebrosidase (GBA1), and UDP-glucose ceramide glucosyltransferase (UGCG) in a sex-balanced MDD cohort. Normalized gene expression was determined by quantitative PCR in patients suffering from MDD (unmedicated n = 63, medicated n = 66) and controls (remitted MDD n = 39, healthy subjects n = 61). We observed that expression levels of SNCA (p = 0.036), GBA1 (p = 0.014), and UGCG (p = 0.0002) were higher in currently depressed patients compared to controls and remitted patients, and expression of GBA1 and UGCG decreased in medicated patients during three weeks of therapy. Additionally, in subgroups, expression was positively correlated with the severity of depression and anxiety. Furthermore, we identified correlations between the gene expression levels and PD-related laboratory parameters. Our findings suggest that SNCA, GBA1, and UGCG analysis could be instrumental in the search for biomarkers of MDD and in understanding the overlapping pathological mechanisms underlying neuro-psychiatric diseases.

GBA1 inactivation in oligodendrocytes affects myelination and induces neurodegenerative hallmarks and lipid dyshomeostasis in mice.

BACKGROUND: Mutations in the ß-glucocerebrosidase (GBA1) gene do cause the lysosomal storage Gaucher disease (GD) and are among the most frequent genetic risk factors for Parkinson's disease (PD). So far, studies on both neuronopathic GD and PD primarily focused on neuronal manifestations, besides the evaluation of microglial and astrocyte implication. White matter alterations were described in the central nervous system of paediatric type 1 GD patients and were suggested to sustain or even play a role in the PD process, although the contribution of oligodendrocytes has been so far scarcely investigated. METHODS: We exploited a system to study the induction of central myelination in vitro, consisting of Oli-neu cells treated with dibutyryl-cAMP, in order to evaluate the expression levels and function of ß-glucocerebrosidase during oligodendrocyte differentiation. Conduritol-B-epoxide, a ß-glucocerebrosidase irreversible inhibitor was used to dissect the impact of ß-glucocerebrosidase inactivation in the process of myelination, lysosomal degradation and α-synuclein accumulation in vitro. Moreover, to study the role of ß-glucocerebrosidase in the white matter in vivo, we developed a novel mouse transgenic line in which ß-glucocerebrosidase function is abolished in myelinating glia, by crossing the Cnp1-cre mouse line with a line bearing loxP sequences flanking Gba1 exons 9-11, encoding for ß-glucocerebrosidase catalytic domain. Immunofluorescence, western blot and lipidomic analyses were performed in brain samples from wild-type and knockout animals in order to assess the impact of genetic inactivation of ß-glucocerebrosidase on myelination and on the onset of early neurodegenerative hallmarks, together with differentiation analysis in primary oligodendrocyte cultures. RESULTS: Here we show that ß-glucocerebrosidase inactivation in oligodendrocytes induces lysosomal dysfunction and inhibits myelination in vitro. Moreover, oligodendrocyte-specific ß-glucocerebrosidase loss-of-function was sufficient to induce in vivo demyelination and early neurodegenerative hallmarks, including axonal degeneration, α-synuclein accumulation and astrogliosis, together with brain lipid dyshomeostasis and functional impairment. CONCLUSIONS: Our study sheds light on the contribution of oligodendrocytes in GBA1-related diseases and supports the need for better characterizing oligodendrocytes as actors playing a role in neurodegenerative diseases, also pointing at them as potential novel targets to set a brake to disease progression.

Gaucher Disease: A Glance from a Medicinal Chemistry Perspective.

Rare diseases are particular pathological conditions affecting a limited number of people and few drugs are known to be effective as therapeutic treatment. Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, belongs to this class of disorders, and it is considered the most common among the Lysosomal Storage Diseases. The two main therapeutic approaches are the Enzyme Replacement Therapy (ERT) and the Substrate Reduction Therapy (SRT). ERT, consisting in replacing the defective enzyme by administering a recombinant enzyme, is effective in alleviating the visceral symptoms, hallmarks of the most common subtype of the disease whereas it has no effects when symptoms involve CNS, since the recombinant protein is unable to significantly cross the Blood Brain Barrier. The SRT strategy involves inhibiting glucosylceramide synthase (GCS), the enzyme responsible for the production of the associated storage molecule. The rational design of new inhibitors of GCS has been hampered by the lack of either the crystal structure of the enzyme or an in-silico model of the active site which could provide important information regarding the interactions of potential inhibitors with the target, but, despite this, interesting results have been obtained and are herein reviewed.

Identification of ß-Glucocerebrosidase Activators for Glucosylceramide hydrolysis.

Several novel chemical series were identified that modulate glucocerebrosidase (GCase). Compounds from these series are active on glucosylceramide, unlike other known GCase modulators. We obtained GCase crystal structures with two compounds that have distinct chemotypes. Positive allosteric modulators bind to a site on GCase and induce conformational changes, but also induce an equilibrium state between monomer and dimer.

Reduced progranulin increases tau and α-synuclein inclusions and alters mouse tauopathy phenotypes via glucocerebrosidase.

Comorbid proteinopathies are observed in many neurodegenerative disorders including Alzheimer's disease (AD), increase with age, and influence clinical outcomes, yet the mechanisms remain ill-defined. Here, we show that reduction of progranulin (PGRN), a lysosomal protein associated with TDP-43 proteinopathy, also increases tau inclusions, causes concomitant accumulation of α-synuclein and worsens mortality and disinhibited behaviors in tauopathy mice. The increased inclusions paradoxically protect against spatial memory deficit and hippocampal neurodegeneration. PGRN reduction in male tauopathy attenuates activity of ß-glucocerebrosidase (GCase), a protein previously associated with synucleinopathy, while increasing glucosylceramide (GlcCer)-positive tau inclusions. In neuronal culture, GCase inhibition enhances tau aggregation induced by AD-tau. Furthermore, purified GlcCer directly promotes tau aggregation in vitro. Neurofibrillary tangles in human tauopathies are also GlcCer-immunoreactive. Thus, in addition to TDP-43, PGRN regulates tau- and synucleinopathies via GCase and GlcCer. A lysosomal PGRN-GCase pathway may be a common therapeutic target for age-related comorbid proteinopathies.

Mitochondrial dysfunction in NPC1-deficiency is not rescued by drugs targeting the glucosylceramidase GBA2 and the cholesterol-binding proteins TSPO and StARD1.

Niemann-Pick type C disease (NPCD) is a rare neurodegenerative disorder most commonly caused by mutations in the lysosomal protein Niemann-Pick C1 (NPC1), which is implicated in cholesterol export. Mitochondrial insufficiency forms a significant feature of the pathology of this disease, yet studies attempting to address this are rare. The working hypothesis is that mitochondria become overloaded with cholesterol which renders them dysfunctional. We examined two potential protein targets-translocator protein (TSPO) and steroidogenic acute regulatory protein D1 (StARD1)-which are implicated in cholesterol transport to mitochondria, in addition to glucocerbrosidase 2 (GBA2), the target of miglustat, which is currently the only approved treatment for NPCD. However, inhibiting these proteins did not correct the mitochondrial defect in NPC1-deficient cells.

A Comparative Biochemical and Pathological Evaluation of Brain Samples from Knock-In Murine Models of Gaucher Disease.

Gaucher disease (GD) is a lysosomal storage disorder stemming from biallelic mutations in GBA1, characterized by glucocerebrosidase dysfunction and glucocerebroside and glucosylsphingosine accumulation. Since phenotypes of murine models of GD often differ from those in patients, the careful characterization of Gba1 mutant mice is necessary to establish their ability to model GD. We performed side-by-side comparative biochemical and pathologic analyses of four murine Gba1 models with genotypes L444P/L444P (p.L483P/p.L483P), L444P/null, D409H/D409H (p.D448H/p.D448H) and D409H/null, along with matched wildtype mice, all with the same genetic background and cage conditions. All mutant mice exhibited significantly lower glucocerebrosidase activity (p < 0.0001) and higher glucosylsphingosine levels than wildtype, with the lowest glucocerebrosidase and the highest glucosylsphingosine levels in mice carrying a null allele. Although glucocerebrosidase activity in L444P and D409H mice was similar, D409H mice showed more lipid accumulation. No Gaucher or storage-like cells were detected in any of the Gba1 mutant mice. Quantification of neuroinflammation, dopaminergic neuronal loss, alpha-synuclein levels and motor behavior revealed no significant findings, even in aged animals. Thus, while the models may have utility for testing the effect of different therapies on enzymatic activity, they did not recapitulate the pathological phenotype of patients with GD, and better models are needed.

The Effect of p.G2019S Mutation in the LRRK2 Gene on the Activity of Lysosomal Hydrolases and the Clinical Features of Parkinson's Disease Associated with p.N370S Mutation in the GBA1 Gene.

BACKGROUND: Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). METHODS: One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. RESULTS: Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). CONCLUSIONS: Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.

Loss of Lipid Carrier ApoE Exacerbates Brain Glial and Inflammatory Responses after Lysosomal GBA1 Inhibition.

Tightly regulated and highly adaptive lipid metabolic and transport pathways are critical to maintaining brain cellular lipid homeostasis and responding to lipid and inflammatory stress to preserve brain function and health. Deficits in the lipid handling genes APOE and GBA1 are the most significant genetic risk factors for Lewy body dementia and related dementia syndromes. Parkinson's disease patients who carry both APOE4 and GBA1 variants have accelerated cognitive decline compared to single variant carriers. To investigate functional interactions between brain ApoE and GBA1, in vivo GBA1 inhibition was tested in WT versus ApoE-deficient mice. The experiments demonstrated glycolipid stress caused by GBA1 inhibition in WT mice induced ApoE expression in several brain regions associated with movement and dementia disorders. The absence of ApoE in ApoE-KO mice amplified complement C1q elevations, reactive microgliosis and astrocytosis after glycolipid stress. Mechanistically, GBA1 inhibition triggered increases in cell surface and intracellular lipid transporters ABCA1 and NPC1, respectively. Interestingly, the absence of NPC1 in mice also triggered elevations of brain ApoE levels. These new data show that brain ApoE, GBA1 and NPC1 functions are interconnected in vivo, and that the removal or reduction of ApoE would likely be detrimental to brain function. These results provide important insights into brain ApoE adaptive responses to increased lipid loads.

Establishment of MUi030-A: A human induced pluripotent stem cell line carrying homozygous L444P mutation in the GBA1 gene to study type-3 Gaucher disease.

Gaucher disease (GD) is a common lysosomal storage disease resulting from mutations in the glucocerebrosidase (GBA1) gene. This genetic disorder manifests with symptoms affecting multiple organs, yet the underlying mechanisms leading to pathology remain elusive. In this study, we successfully generated the MUi030-A human induced pluripotent stem cell (hiPSC) line using a non-integration method from a male type-3 GD patient with a homozygous c.1448T>C (L444P) mutation. These hiPSCs displayed a normal karyotype and pluripotency markers and the remarkable ability to differentiate into cells representing all three germ layers. This resourceful model holds significant promise for illuminating GD's underlying pathogenesis.

Axonal Transport of Lysosomes Is Unaffected in Glucocerebrosidase-Inhibited iPSC-Derived Forebrain Neurons.

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.

A Fixable Fluorescence-Quenched Substrate for Quantitation of Lysosomal Glucocerebrosidase Activity in Both Live and Fixed Cells.

Fluorogenic substrates are emerging tools that enable studying enzymatic processes within their native cellular environments. However, fluorogenic substrates that function within live cells are generally incompatible with cellular fixation, preventing their tandem application with fundamental cell biology methods such as immunocytochemistry. Here we report a simple approach to enable the chemical fixation of a dark-to-light substrate, LysoFix-GBA, which enables quantification of glucocerebrosidase (GCase) activity in both live and fixed cells. LysoFix-GBA enables measuring responses to both chemical and genetic perturbations to lysosomal GCase activity. Further, LysoFix-GBA permits simple multiplexed co-localization studies of GCase activity with subcellular protein markers. This tool will aid studying the role of GCase activity in Parkinson's Disease, creating new therapeutic approaches targeting the GCase pathway. This approach also lays the foundation for an approach to create fixable substrates for other lysosomal enzymes.

Whole Transcriptome Analysis of Substantia Nigra in Mice with MPTP-Induced Parkinsonism Bearing Defective Glucocerebrosidase Activity.

Mutations in the GBA1 gene represent the major genetic risk factor for Parkinson's disease (PD). The lysosomal enzyme beta-glucocerebrosidase (GCase) encoded by the GBA1 gene participates in both the endolysosomal pathway and the immune response. Disruption of these mechanisms is involved in PD pathogenesis. However, molecular mechanisms of PD associated with GBA1 mutations (GBA-PD) are unknown today in particular due to the partial penetrance of GBA1 variants in PD. The modifiers of GBA1 penetrance have not been elucidated. We characterized the transcriptomic profiles of cells from the substantia nigra (SN) of mice with co-injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and selective inhibitor of GCase activity (conduritol-ß-epoxide, (CBE)) to mimic PD bearing GCase dysfunction (MPTP+CBE), mice treated with MPTP, mice treated with CBE and control mice treated with injection of sodium chloride (NaCl) (vehicle). Differential expression analysis, pathway enrichment analysis, and outlier detection were performed. Functional clustering of differentially represented transcripts revealed more processes associated with the functioning of neurogenesis, inflammation, apoptosis and autophagy in MPTP+CBE and MPTP mice than in vehicle mice, with a more pronounced alteration of autophagy processes in MPTP+CBE mice than in MPTP mice. The PI3K-Akt-mTOR signaling pathway may be considered a potential target for therapy in PD with GCase dysfunction.

LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson's disease.

Last data demonstrated that exonic variants of LRRK2 (p.G2019S, p.M1646T) may affect the catalytic activity of lysosomal enzyme glucocerebrosidase (GCase) probably through the phosphorylation of Rab10 protein. We aimed to evaluate an association of LRRK2 exonic variants previously associated with alteration of phosphorylation levels for Rab10Thr73 with PD risk in Russian population and analyze an impact of p.G2019S mutation and selected LRRK2 variants on lysosomal hydrolase activities. LRRK2 variants were determined by full sequencing of LRRK2 in 508 PD patients and 470 controls from Russian population. Activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase A (GLA), acid sphingomyelinase (ASMase) and concentrations of their corresponded substrates (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM), respectively) were estimated in 211 PD patients and 179 controls by liquid chromatography with tandem mass spectrometry (LC-MS-MS) in dry blood spots. p.M1646T and p.N2081D were associated with PD (OR = 2.33, CI 95%: 1.1215 to 4.8253, p = 0.023; OR = 1.89, 95%CI: 1.0727 to 3.3313, p = 0.028, respectively) in Russian population. An increased LysoGb3 concentration was found in p.G2019S and p.N2081D LRRK2 carriers among PD patients compared to both PD patients and controls (p.G2019S: p = 0.00086, p = 0.0004, respectively; p.N2081D: p = 0.012, p = 0.0076, respectively). A decreased ASMase activity in p.G2019S LRRK2 carriers among PD patients (p = 0.014) was demonstrated as well. Our study supported possible involvement of LRRK2 dysfunction in an alteration of sphingolipid metabolism in PD.

Restoration of ß-GC trafficking improves the lysosome function in Gaucher disease.

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low ß-glucocerebrosidase (ß-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant ß-GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based ß-GC activity assay followed by reporter trafficking assay utilizing ß-GC-mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the ß-GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their ß-GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD.