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Gaucher disease (GD) is caused by biallelic GBA1/Gba1 mutations that encode defective glucocerebrosidase (GCase). Progranulin (PGRN, encoded by GRN/Grn) is a modifier of GCase, but the interplay between PGRN and GCase, specifically GBA1/Gba1 mutations, contributing to GD severity is unclear. Mouse models were developed with various dosages of Gba1 D409V mutation against the PGRN deficiency (Grn-/-) [Grn-/-;Gba1D409V/WT (PG9Vwt), Grn-/-;Gba1D409V/D409V (PG9V), Grn-/-;Gba1D409V/Null (PG9VN)]. Disease progression in those mouse models was characterized by biochemical, pathological, transcriptomic, and neurobehavioral analyses. Compared to PG9Vwt, Grn-/-;Gba1WT/Null and Grn-/- mice that had a higher level of GCase activity and undetectable pathologies, homozygous or hemizygous D409V in PG9V or PG9VN, respectively, resulted in profound inflammation and neurodegeneration. PG9VN mice exhibited much earlier onset, shorter life span, tissue fibrosis, and more severe phenotypes than PG9V mice. Glycosphingolipid accumulation, inflammatory responses, lysosomal-autophagy dysfunction, microgliosis, retinal gliosis, as well as α-Synuclein increases were much more pronounced in PG9VN mice. Neurodegeneration in PG9VN was characterized by activated microglial phagocytosis of impaired neurons and programmed cell death due to necrosis and, possibly, pyroptosis. Brain transcriptomic analyses revealed the intrinsic relationship between D409V dosage, and the degree of altered gene expression related to lysosome dysfunction, microgliosis, and neurodegeneration in GD, suggesting the disease severity is dependent on a GCase activity threshold related to Gba1 D409V dosage and loss of PGRN. These findings contribute to a deeper understanding of GD pathogenesis by elucidating additional underlying mechanisms of interplay between PGRN and Gba1 mutation dosage in modulating GCase function and disease severity in GD and GBA1-associated neurodegenerative diseases.
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Targeted delivery of α-synuclein using AAV vectors has over the two decades since its introduction developed into a versatile tool for modeling different aspects of synucleinopathy, mimicking those seen in Parkinson's disease and related Lewy body disorders. The viral vector approach to disease modeling is attractive in that the expression of α-synuclein, wild-type or mutated, can be confined to defined anatomical structures and targeted to selected cell populations using either cell-type specific promoter constructs or different natural or engineered AAV serotypes. AAV-α-synuclein was initially used to model progressive α-synuclein pathology in nigral dopamine neurons, and, like the standard 6-OHDA model, it has most commonly been applied unilaterally, using the non-injected side as a reference and control. In recent years, however, the AAV-α-synuclein model has become more widely used to induce Parkinson-like synuclein pathology in other relevant neuronal systems, such as the brainstem noradrenergic and serotonergic neurons, the vagal motor neurons, as well as in oligodendrocytes, the prime target relevant to the pathology seen in multiple system atrophy. The purpose of this review is to give an overview of the progress made in the use of the AAV-α-synuclein model over the last two decades and summarize the state-of-the art in the use of the AAV-α-synuclein model for disease modeling in rats and mice.
Misfolding of the neuronal protein α-synuclein is central to the cellular processes that underlie the development of Parkinson's disease and related disorders, such as dementia with Lewy bodies and multiple system atrophy. Targeted delivery of α-synuclein using adeno-associated virus, AAV, has become a standard tool to model the disease process in animals. This AAV-α-synuclein model of Parkinson's disease was introduced two decades ago and over the ensuing decades it has become a widely used standard tool for experimental studies in animals. The usefulness of the AAV-α-synuclein model is largely due to its flexibility and versatility as an experimental tool. In this review the authors summarize the state-of-the art in this field and review the range of applications that has been developed using AAV-α-synuclein alone, in single hit models, or in combinations with other interacting risk factors, in double hit models.
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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.
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Doença de Gaucher , Glucosilceramidase , Proteínas de Membrana Lisossomal , Receptores Depuradores , Saposinas , Glucosilceramidase/genética , Glucosilceramidase/deficiência , Glucosilceramidase/metabolismo , Humanos , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Saposinas/deficiência , Saposinas/genética , Saposinas/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/genética , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Fibroblastos/metabolismo , Mutação , Lisossomos/metabolismo , Lisossomos/enzimologia , Hexosaminidases/metabolismo , Hexosaminidases/genética , Hexosaminidases/deficiência , Masculino , FemininoRESUMO
INTRODUCTION: Gaucher's disease (GD) is caused by biallelic mutations in the GBA1 gene, leading to reduced glucocerebrosidase (GCase) activity and substrate (glucosylceramide and glucosylsphingosine, GlcSph) accumulation. GBA1 variant carriers are at risk of Parkinson's disease (PD), but only those with biallelic mutations cross the threshold of GCase reduction, leading to substrate accumulation and GD. The link between GBA1 mutations, GD and PD is not fully understood. Here we aimed at reporting the results of a large PD population screening with dried blood spot tests for GD. METHODS: We measured GCase activity and GlcSph levels in 1344 PD patients with dried blood spot tests, and performed GBA1 genetic sequencing. RESULTS: While the GCase activity was reduced in GBA1-PD carriers compared to wild type PD, GlcSph was increased in GBA1-PD compared to GBA1-controls, regardless of the underlying type of GBA1 variant. 13.6 % and 0.4 % of PD patients had mono- or biallelic GBA1 mutations respectively. GCase deficiency, lipid accumulation and clinical manifestations of GD was detected in five PD patients with biallelic GBA1 mutations, of whom four had a risk combined with a GD causing variant. CONCLUSIONS: GlcSph appearing higher in PD may represent a reliable biomarker of the disease and deserves to be further investigated. This study highlights the importance of screening PD patients for possible underlying GD, which is a treatable condition that should not be missed. We diagnosed GD cases carrying a "risk" variant in one allele, which is an unprecedented finding deserving further investigation.
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Doença de Gaucher , Glucosilceramidase , Doença de Parkinson , Psicosina , Humanos , Glucosilceramidase/genética , Doença de Gaucher/genética , Doença de Gaucher/sangue , Doença de Parkinson/genética , Doença de Parkinson/sangue , Psicosina/análogos & derivados , Psicosina/sangue , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Mutação , Teste em Amostras de Sangue Seco , Adulto , Idoso de 80 Anos ou maisRESUMO
The conventional approach to developing light-sensitive glycosidase activity regulators, involving the combination of a glycomimetic moiety and a photoactive azobenzene module, results in conjugates with differences in glycosidase inhibitory activity between the interchangeable E and Z-isomers at the azo group that are generally below one-order of magnitude. In this study, we have exploited the chemical mimic character of sp2-iminosugars to access photoswitchable p- and o-azobenzene α-O-glycosides based on the gluco-configured representative ONJ. Notably, we achieved remarkably high switching factors for glycosidase inhibition, favoring either the E- or Z-isomer depending on the aglycone structure. Our data also indicate a correlation between the isomeric state of the azobenzene module and the selectivity towards α- and ß-glucosidase isoenzymes. The most effective derivative reached over a 103-fold higher inhibitory potency towards human ß-glucocerebrosidase in the Z as compared with the E isomeric form. This sharp contrast is compatible with ex-vivo activation and programmed self-deactivation at physiological temperatures, positioning it as a prime candidate for pharmacological chaperone therapy in Gaucher disease. Additionally, our results illustrate that chemical tailoring enables the engineering of photocommutators with the ability to toggle inhibition between α- and ß-glucosidase enzymes in a reversible manner, thus expanding the versatility and potential therapeutic applications of this approach.
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Compostos Azo , Inibidores Enzimáticos , Glicosídeo Hidrolases , Glicosídeos , Imino Açúcares , Humanos , Compostos Azo/química , Compostos Azo/farmacologia , Compostos Azo/síntese química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosídeos/farmacologia , Glicosídeos/síntese química , Imino Açúcares/química , Imino Açúcares/farmacologia , Imino Açúcares/síntese química , Luz , Estrutura Molecular , Relação Estrutura-Atividade , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Glucosilceramidase/farmacologiaRESUMO
One of the most common genetic risk factors for Parkinson's disease (PD) are variants in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GCase deficiency has been associated with an increased PD risk, but not all individuals with low GCase activity are carriers of GBA1 mutations, suggesting other factors may be acting as modifiers. We aimed to discover common variants associated with GCase activity, as well as replicate previously reported associations, by performing a genome-wide association study using two independent cohorts: a Columbia University cohort consisting of 697 PD cases and 347 controls and the Parkinson's Progression Markers Initiative (PPMI) cohort consisting of 357 PD cases and 163 controls. As expected, GBA1 variants have the strongest association with decreased activity, led by p.N370S (beta = -4.36, se = 0.32, p = 5.05e-43). We also identify a novel association in the GAA locus (encoding for acid alpha-glucosidase, beta = -0.96, se = 0.17, p = 5.23e-09) that may be the result of an interaction between GCase and acid alpha-glucosidase based on various interaction analyses. Lastly, we show that several PD-risk loci are potentially associated with GCase activity. Further research will be needed to replicate and validate our findings and to uncover the functional connection between acid alpha-glucosidase and GCase.
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OBJECTIVE: Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). METHODS: Additionally to cerebral MRI, protein structural analyses of the ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. RESULTS: Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. INTERPRETATION: The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.
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Catepsina B , Catepsina D , Galactosilceramidase , Leucodistrofia de Células Globoides , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/diagnóstico , Masculino , Catepsina D/genética , Catepsina D/metabolismo , Galactosilceramidase/genética , Adulto , Catepsina B/genética , Catepsina B/metabolismo , Paraplegia/genética , Idade de Início , Glucosilceramidase/genética , Lisossomos , Fibroblastos/metabolismo , Fibroblastos/patologiaRESUMO
Gaucher disease (GD) stands as one of the most prevalent lysosomal disorders, yet neuronopathic GD (nGD) is an uncommon subset characterized by a wide array of clinical manifestations that complicate diagnosis, particularly when neurological symptoms are understated. nGD may manifest as the acute neuronopathic type, or GD type 2 (GD2), either prenatally or within the first weeks to months of life, whereas GD type 3 (GD3) symptoms may emerge at any point during childhood or occasionally in adolescence. The clinical presentation encompasses severe systemic involvement to mild visceral disease, often coupled with a spectrum of progressive neurological signs and symptoms such as cognitive impairment, ataxia, seizures, myoclonus, varying degrees of brainstem dysfunction presenting with stridor, apneic episodes, and/or impaired swallowing. This manuscript aims to provide a comprehensive review of the incidence, distinctive presentations, and diverse clinical phenotypes of nGD across various countries and regions. It will explore the natural history of the neurodegenerative process in GD, shedding light on its various manifestations during infancy and childhood, and offer insights into the diagnostic journey, the challenges faced in the clinical management, and current and investigative therapeutic approaches for GD's neurological variants.
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Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small pro-luminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and non-inhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: the fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 provided direct visualization of GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy, by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically-relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of new chemical matter targeting GCase, ultimately leading to a viable therapeutic for two protein-misfolding diseases.
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BACKGROUND: To date, no disease modifying therapies are available for Parkinson's disease (PD). Since PD is the second most prevalent neurodegenerative disorder, there is a high demand for such therapies. Both environmental and genetic risk factors play an important role in the etiology and progression of PD. The most common genetic risk factor for PD is a mutation in the GBA1(GBA)-gene, encoding the lysosomal enzyme glucocerebrosidase (GCase). The mucolytic ambroxol is a repurposed drug, which has shown the property to upregulate GCase activity in-vitro and in-vivo. Ambroxol therefore has the potency to become a disease modifying therapy in PD, which was the reason to design this randomized controlled trial with ambroxol in PD patients. METHODS: This trial is a single-center, double-blind, randomized, placebo-controlled study, including 80 PD patients with a GBA mutation, receiving either ambroxol 1800 mg/day or placebo for 48 weeks. The primary outcome measure is the Unified Parkinson's Disease Rating Scale motor subscore (part III) of the Movement Disorder Society (MDS-UPDRSIII) in the practically defined off-state at 60 weeks (after a 12-week washout period). Secondary outcomes include a 3,4-dihydroxy-6-18F-fluoro-I-phenylalanine ([18F]FDOPA) PET-scan of the brain, Magnetic Resonance Imaging (with resting state f-MRI and Diffusion Tensor Imaging), GCase activity, both intra- and extracellularly, sphingolipid profiles in plasma, Montreal Cognitive Assessment (MoCA), quality of life (QoL) measured by the Parkinson's Disease Questionnaire (PDQ-39) and the Non-Motor Symptom Scale (NMSS) questionnaire. DISCUSSION: Ambroxol up to 1200 mg/day has shown effects on human cerebrospinal fluid endpoints, which supports at least passage of the blood-brain-barrier. The dose titration in this trial up to 1800 mg/day will reveal if this dose level is safe and also effective in modifying the course of the disease. TRIAL REGISTRATION: NCT05830396. Registration date: March 20, 2023.
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Ambroxol , Glucosilceramidase , Mutação , Doença de Parkinson , Humanos , Ambroxol/administração & dosagem , Ambroxol/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/diagnóstico por imagem , Glucosilceramidase/genética , Método Duplo-Cego , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Resultado do Tratamento , Expectorantes/uso terapêutico , Expectorantes/administração & dosagem , AdultoRESUMO
Background: Gaucher disease is a lysosomal storage disorder caused by functional glucocerebrosidase enzyme deficiency. Hepatosplenomegaly and hematological complications are found in both Gaucher disease and Acid Sphingomyelinase Deficiency, which is caused by acid sphingomyelinase dysfunction. The possible overlap in clinical presentation can cause diagnostic errors in differential diagnosis. For this reason, in patients with an initial clinical suspicion of Gaucher disease, we aimed to carry out a parallel screening of acid sphingomyelinase and glucocerebrosidase. Methods: Peripheral blood samples of 627 patients were collected, and enzymatic activity analysis was performed on both glucocerebrosidase and acid sphingomyelinase. The specific gene was studied in samples with null or reduced enzymatic activity. Specific molecular biomarkers helped to achieve the correct diagnosis. Results: In 98.7% of patients, normal values of glucocerebrosidase activity excluded Gaucher disease. In 8 of 627 patients (1.3%), the glucocerebrosidase enzymatic activity assay was below the normal range, so genetic GBA1 analysis confirmed the enzymatic defect. Three patients (0.5%) had normal glucocerebrosidase activity, so they were not affected by Gaucher disease, and showed decreased acid sphingomyelinase activity. SMPD1 gene mutations responsible for Acid Sphingomyelinase Deficiency were found. The levels of specific biomarkers found in these patients further strengthened the genetic data. Conclusions: Our results suggest that in the presence of typical signs and symptoms of Gaucher disease, Acid Sphingomyelinase Deficiency should be considered. For this reason, the presence of hepatosplenomegaly, thrombocytopenia, leukocytopenia, and anemia should alert clinicians to analyze both enzymes by a combined screening. Today, enzyme replacement therapy is available for the treatment of both pathologies; therefore, prompt diagnosis is essential for patients to start accurate treatment and to avoid diagnostic delay.
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Parkinson's disease (PD) is a common neurodegenerative disease with progressive loss of dopaminergic neurons in substantia nigra and the presence of α-synuclein-immunoreactive inclusions. Gaucher's disease is caused by homozygous mutations in ß-glucocerebrosidase gene (GBA). GBA mutation carriers have an increased risk of PD. Coptis chinensis (C. chinensis) rhizome extract is a major herb widely used to treat human diseases. This study examined the association of GBA L444P mutation with Taiwanese PD in 1016 cases and 539 controls. In addition, the protective effects of C. chinensis rhizome extract and its active constituents (berberine, coptisine, and palmatine) against PD were assayed using GBA reporter cells, LC3 reporter cells, and cells expressing mutated (A53T) α-synuclein. Case-control study revealed that GBA L444P carriers had a 3.93-fold increased risk of PD (95% confidence interval (CI): 1.37-11.24, p = 0.006) compared to normal controls. Both C. chinensis rhizome extract and its constituents exhibited chemical chaperone activity to reduce α-synuclein aggregation. Promoter reporter and endogenous GBA protein analyses revealed that C. chinensis rhizome extract and its constituents upregulated GBA expression in 293 cells. In addition, C. chinensis rhizome extract and its constituents induced autophagy in DsRed-LC3-expressing 293 cells. In SH-SY5Y cells expressing A53T α-synuclein, C. chinensis rhizome extract and its constituents reduced α-synuclein aggregation and associated neurotoxicity by upregulating GBA expression and activating autophagy. The results of reducing α-synuclein aggregation, enhancing GBA expression and autophagy, and protecting against α-synuclein neurotoxicity open up the therapeutic potentials of C. chinensis rhizome extract and constituents for PD.
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Berberina , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Berberina/análogos & derivados , Estudos de Casos e Controles , Coptis chinensis , Neurônios Dopaminérgicos/metabolismo , Mutação , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , RizomaRESUMO
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.
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Doença de Gaucher , Glucosilceramidase , Doença de Parkinson , Humanos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Células HEK293 , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/genética , Lisossomos/metabolismo , Receptores Depuradores/genética , Receptores Depuradores/metabolismoRESUMO
Gaucher disease (GD) is a lysosomal storage disorder caused by a deficiency in the GBA1-encoded enzyme, ß-glucocerebrosidase. Enzyme replacement therapy is ineffective for neuronopathic Gaucher disease (nGD). High-dose ambroxol has been administered as an alternative treatment for a group of patients with nGD. However, little is known about the clinical indication and the long-term outcome of patients after ambroxol therapy. We herein report a case of a female patient who presented with a progressive disease of GD type 2 from 11 months of age and had the pathogenic variants of p.L483P (formerly defined as p.L444P) and p.R502H (p.R463H) in GBA1. A combined treatment of imiglucerase with ambroxol started improving the patient's motor activity in 1 week, while it kept the long-lasting effect of preventing the deteriorating phenotype for 30 months. A literature review identified 40 patients with nGD, who had received high-dose ambroxol therapy. More than 65% of these patients favorably responded to the molecular chaperone therapy, irrespective of p.L483P homozygous, heterozygous or the other genotypes. These results highlight the long-lasting effect of ambroxol-based chaperone therapy for patients with an expanding spectrum of mutations in GBA1.
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Ambroxol , Doença de Gaucher , Doenças por Armazenamento dos Lisossomos , Humanos , Feminino , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Gaucher/patologia , Ambroxol/uso terapêutico , Terapia Combinada , Chaperonas MolecularesRESUMO
A concise asymmetric synthesis of clickable enantiomeric pyrrolidines was achieved using Crabbé-Ma allenation. The synthesized iminosugars were grafted by copper-free strain-promoted alkyne-azide cycloaddition onto phosphorus dendrimers. The hexavalent and dodecavalent pyrrolidines were evaluated as ß-glucocerebrosidase inhibitors. The level of inhibition suggests that monofluorocyclooctatriazole group may contribute to the affinity for the protein leading to potent multivalent inhibitors. Docking studies were carried out to rationalize these results. Then, the iminosugars clusters were evaluated as pharmacological chaperones in Gaucher patients' fibroblasts. An increase in ß-glucocerebrosidase activity was observed with hexavalent and dodecavalent pyrrolidines at concentrations as low as 1 µM and 0.1 µM, respectively. These iminosugar clusters constitute the first example of multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.
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Doença de Gaucher , Imino Açúcares , Humanos , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase , Pirrolidinas/farmacologia , Inibidores Enzimáticos/farmacologiaRESUMO
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.
Assuntos
Doença de Gaucher , Doença de Parkinson , Animais , Camundongos , alfa-Sinucleína/metabolismo , Animais Geneticamente Modificados/metabolismo , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Lipídeos , Mutação , Doença de Parkinson/metabolismoRESUMO
BACKGROUND: SNCA p.V15A was reported in five families. In vitro models showed increased aggregation and seeding activity, mitochondrial damage, and apoptosis. Mutant flies had reduced flying ability and survival. OBJECTIVES: To clinically and functionally evaluate SNCA p.V15A in a large Italian family with Parkinson's disease (PD). METHODS: Genetic diagnosis was reached through next-generation sequencing. Pathogenicity was assessed by molecular dynamics simulation and biochemical studies on peripheral blood mononuclear cells (PBMCs). RESULTS: Five siblings carried SNCA p.V15A; three developed bradykinetic-rigid PD in their 50s with rapid motor progression and variable cognitive impairment. A fourth sibling had isolated mood disturbance, whereas the fifth was still unaffected at age 47. The mutant protein showed decreased stability and an unstable folded structure. Proband's PBMCs showed elevated total and phosphorylated α-synuclein (α-syn) levels and significantly reduced glucocerebrosidase activity. CONCLUSION: This study demonstrates accumulation of α-synV15A in PBMCs and strengthens the link between α-syn pathophysiology and glucocerebrosidase dysfunction. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Glucosilceramidase , Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Glucosilceramidase/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Masculino , Pessoa de Meia-Idade , Feminino , Leucócitos Mononucleares/metabolismo , Linhagem , Mutação/genética , IdosoRESUMO
Hexaconazole is a known fungicide for agricultural purposes. It has bioaccumulation ability which makes it important for its toxicological characterization. There are various neurological impacts of pollutants on human health. Therefore, in this study, we have done predictive analyses of the interaction mechanism of hexaconazole by molecular interaction analysis, molecular dynamics simulation, and Poisson-Boltzmann surface area (MM-PBSA) to assess hexaconazole's potency to disrupt the homeostasis of glucocerebrosidase (-7.9 kcal/mol) and parkin (-5.67 kcal/mol) proteins which have significant roles in the manifestation of Parkinson disease. The findings reveal that hexaconazole has the potency to form stable interactions with glucocerebrosidase and parkin. This research provides a molecular and atomic-level understanding of how hexaconazole exposure may disrupt the homeostasis of glucocerebrosidase and parkin. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration, and hydrogen bonding exhibited the potent molecular interactions of hexaconazole, which may lead to neurological manifestations such as Parkinson disease.
RESUMO
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.
Assuntos
Doença de Gaucher , Psicosina/análogos & derivados , Camundongos , Humanos , Animais , Idoso , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Modelos Animais de Doenças , Encéfalo/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , MutaçãoRESUMO
Gaucher disease (GD) is mainly caused by glucocerebrosidase (GCase) enzyme deficiency due to genetic variations in the GBA1 gene leading to the toxic accumulation of sphingolipids in various organs, which causes symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, and neurological manifestations. GD is clinically classified into the non-neuronopathic type 1, and the acute and chronic neuronopathic forms, types 2 and 3, respectively. In addition to the current approved GD medications, the repurposing of Ambroxol (ABX) has emerged as a prospective enzyme enhancement therapy option showing its potential to enhance mutated GCase activity and reduce glucosylceramide accumulation in GD-affected tissues of different GBA1 genotypes. The variability in response to ABX varies across different variants, highlighting the diversity in patients' therapeutic outcomes. Its oral availability and safety profile make it an attractive option, particularly for patients with neurological manifestations. Clinical trials are essential to explore further ABX's potential as a therapeutic medication for GD to encourage pharmaceutical companies' investment in its development. This review highlights the potential of ABX as a pharmacological chaperone therapy for GD and stresses the importance of addressing response variability in clinical studies to improve the management of this rare and complex disorder.