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Moraitou, Marina, Georgios Hadjigeorgiou, Ioannis Monopolis, Efthimios Dardiotis, Maria Bozi, Demitris Vassilatis, Lluisa Vilageliu, et al. "β-Glucocerebrosidase gene mutations in two cohorts of Greek patients with sporadic Parkinson's disease." Molecular Genetics and Metabolism 104, no. 1-2 (September 2011): 149–52. http://dx.doi.org/10.1016/j.ymgme.2011.06.015.
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Chen, Mingyi, and Jun Wang. "Gaucher Disease: Review of the Literature." Archives of Pathology & Laboratory Medicine 132, no. 5 (May 1, 2008): 851–53. http://dx.doi.org/10.5858/2008-132-851-gdrotl.
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Abstract We present a brief review of Gaucher disease, the most common lysosomal storage disease. Gaucher disease is a rare autosomal recessive disorder characterized by defective function of the catabolic enzyme β-glucocerebrosidase, leading to an accumulation of its substrate, glucocerebroside, in the mononuclear phagocyte system, especially histiocytes in the spleen, lymph nodes, and bone marrow; Kupffer cells in the liver; osteoclasts in bone; microglia in the central nervous system; alveolar macrophages in the lungs; and histiocytes in the gastrointestinal tracts, genitourinary tracts, and the peritoneum. Clinical signs and symptoms include neurologic dysfunctions, bone infarcts and malformations, hepatosplenomegaly and hypersplenism leading to anemia, neutropenia, and thrombocytopenia. Enzyme replacement therapy with recombinant glucocerebrosidase is the mainstay of treatment for Gaucher disease, which became the first successfully managed lipid storage disease. Future treatments may include oral enzyme replacement and/or gene therapy interventions.
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Singla, Sanjay, Rameshwar Ninama, Bhupesh Jain, and Suresh Goyal. "Gaucher's disease: a case report." International Journal of Research in Medical Sciences 5, no. 4 (March 28, 2017): 1712. http://dx.doi.org/10.18203/2320-6012.ijrms20171295.
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Gaucher's disease (GD) is an autosomal recessive disorder, characterized by lack of acid β-glucosidase (glucocerebrosidase) enzyme resulting in accumulation of glucosylceramide in different organs. This enzyme is encoded by a gene on chromosome 1. Accumulation of glucosylceramide in tissues leads to multisystem organ involvement viz. liver, spleen, bone marrow, lungs and central nervous system. It is common in Ashkenazi Jews but rare in India. Around five hundred cases are identified and diagnosed in India. Serum β-glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. Here we report a case of Gaucher’s disease.
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Pocovi, Miguel, Ana Cenarro, Fernando Civeira, Miguel A. Torralba, Juan I. Perez-Calvo, Pilar Mozas, Pilar Giraldo, et al. "β-glucocerebrosidase gene locus as a link for Gaucher's disease and familial hypo-α-lipoproteinaemia." Lancet 351, no. 9120 (June 1998): 1919–23. http://dx.doi.org/10.1016/s0140-6736(97)09490-7.
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Laubscher, Kevin H., Robert H. Glew, Robert E. Lee, and Richard T. Okinaka. "Use of denaturing gradient gel electrophoresis to identify mutant sequences in the β-glucocerebrosidase gene." Human Mutation 3, no. 4 (1994): 411–15. http://dx.doi.org/10.1002/humu.1380030418.
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Massaro, Giulia, Michael P. Hughes, Sammie M. Whaler, Kerri-Lee Wallom, David A. Priestman, Frances M. Platt, Simon N. Waddington, and Ahad A. Rahim. "Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes." Human Molecular Genetics 29, no. 12 (January 10, 2020): 1933–49. http://dx.doi.org/10.1093/hmg/ddz317.
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Abstract Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme β-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.
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Radha Rama Devi, Akella, Srilatha Kadali, Ananthaneni Radhika, Vineeta Singh, M. Kumar, Gummadi Reddy, and Shaik Naushad. "Acute Gaucher Disease-Like Condition in an Indian Infant with a Novel Biallelic Mutation in the Prosaposin Gene." Journal of Pediatric Genetics 08, no. 02 (October 26, 2018): 081–85. http://dx.doi.org/10.1055/s-0038-1675372.
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AbstractThis is the first reported case of prosaposin (PSAP) mutation from India manifesting as an acute neuronal Gaucher disease-like condition. A 2-month-old male baby presented with encephalopathy, resistant tonic–clonic seizures, moderate hepatosplenomegaly, hypotonia, and cherry red spot in the retinae. The child had anemia, thrombocytopenia, elevated chitotriosidase, and normal activity of acid sphingomyelinase and low normal activity of β-glucosidase 1 (β-glucocerebrosidase 1, GBA). The child succumbed in the fourth month of life due to persistent respiratory distress and refractory seizures. The clinical phenotype, cherry red spots, elevated chitotriosidase, and lysosomal assays led to the suspicion of Gaucher disease. Exome sequencing revealed a homozygous stop codon mutation in the PSAP gene (c.G1228T, p.Glu410ter). Prenatal diagnosis in the next pregnancy revealed a carrier fetus, who was unaffected postnatally. The diagnosis of specific activator deficiency such as saposin C and saposin D deficiency (in the current study) should be considered and tested for when Gaucher disease is suspected in an infant with partially deficient or near normal GBA activity.
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Naz, Arshi, Qurat Abedin, Shariq Ahmed, Saima Siddiqui, and Tahir Shamsi. "Identification of GBA Gene Mutations in 19 Pakistani Unrelated Patients of Gaucher Disease." Blood 132, Supplement 1 (November 29, 2018): 4952. http://dx.doi.org/10.1182/blood-2018-99-120385.
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Abstract Introduction: Gaucher disease (GD) is one of the lysosomal storage diseases that is rare and inherited autosomal recessively. There is insufficiency of glucocerebrosidase enzyme that leads to the build up of un-degraded substrates in white blood cells causing anemia, hepatosplenomegaly and skeletal disease. This enzyme deficiency is linked with the defect of its gene (GBA) that codes for this enzyme. Initial diagnosis is made by the estimation of glucocerebrosidase level in blood and confirmed by genetic analysis of GBA gene. To identify the mutations of GBA gene in Pakistani patients with GD from different regions of Pakistan. Sampling & methodology: The sample and demographic data was collected in National Institute of Blood Disease and Bone marrow Transplantation after approval of IRB and written informed consent of patients. We collected total 19 blood samples, out of which 5 had Gaucher's disease, 10 samples were parents of the index cases and 4 were control. The methodology consisted of DNA extraction and quantification from peripheral blood. Genetic analysis of coding regions of GBA gene was done via gene amplification, gel electrophoresis and sequencing. Result: Mutation was found in two out of five families that makes the prevalence of GBA gene mutation 40%. These were diagnosed on reduced enzyme levels and found to have L444P (c.1448T>C) mutation in homozygous form in 10th exon of GBA gene. The parents of that patient carried the same mutation in one allele. Rest of the patients who were diagnosed on bone marrow morphology showed no mutation in GBA gene. Conclusion: Our results illustrate that GBA gene mutation was found in those patients who were diagnosed by the estimation of β-glucosidase enzyme levels rather than on bone marrow morphology. In our population, the mutation L444P was found, which is the most frequent gene mutation found in the world. Since this study is conducted in a small number of patients therefore it is recommended that large cohorts of patients should be evaluated in future for genetic mutations among Gaucher's patients in Pakistan Key words: Gaucher disease, storage disorder, GBA gene Disclosures No relevant conflicts of interest to declare.
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Cabasso, Or, Sumit Paul, Gali Maor, Metsada Pasmanik-Chor, Wouter Kallemeijn, Johannes Aerts, and Mia Horowitz. "The Uncovered Function of the Drosophila GBA1a-Encoded Protein." Cells 10, no. 3 (March 12, 2021): 630. http://dx.doi.org/10.3390/cells10030630.
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Human GBA1 encodes lysosomal acid β-glucocerebrosidase (GCase), which hydrolyzes cleavage of the beta-glucosidic linkage of glucosylceramide (GlcCer). Mutations in this gene lead to reduced GCase activity, accumulation of glucosylceramide and glucosylsphingosine, and development of Gaucher disease (GD). Drosophila melanogaster has two GBA1 orthologs. Thus far, GBA1b was documented as a bone fide GCase-encoding gene, while the role of GBA1a encoded protein remained unclear. In the present study, we characterized a mutant variant of the fly GBA1a, which underwent ERAD and mildly activated the UPR machinery. RNA-seq analyses of homozygous mutant flies revealed upregulation of inflammation-associated as well as of cell-cycle related genes and reduction in programmed cell death (PCD)-associated genes, which was confirmed by qRT-PCR. We also observed compromised cell death in the midgut of homozygous larvae and a reduction in pupation. Our results strongly indicated that GBA1a-encoded protein plays a role in midgut maturation during larvae development.
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Karakoyun, Miray, Ebru Canda, Ezgi Kiran Tasci, Eser Dogan, Mahmut Coker, and Sema Aydogdu. "Two siblings with Gaucher type 3c: different clinical presentations." Journal of Pediatric Endocrinology and Metabolism 32, no. 5 (May 27, 2019): 533–36. http://dx.doi.org/10.1515/jpem-2018-0549.
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Abstract Background Gaucher disease (GD) is a lysosomal storage disorder caused by autosomal recessive mutations in the glucocerebrosidase (GBA) gene, which encodes acid β-glucosidase. GD type 3c is a rare group characterised by cardiovascular involvement, and homozygous D448H is the most frequent mutation. Case presentation We describe two patients who had homozygous D448H mutations. The index patient had hepatosplenomegaly, liver insufficiency and cardiac involvement and her sister had severe cardiac involvement with cardiomyopathy and diffuse aortic calcification. The index case’s liver was transplanted at the age of 6 months from a related donor and her sister who had severe cardiovascular disease died at the age of 12 years. Conclusions Our patients had clinical variability. We need to discuss whether liver involvement could be the initial signs in patients with GD type 3c.
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Zunke, Friederike, Lisa Andresen, Sophia Wesseler, Johann Groth, Philipp Arnold, Michelle Rothaug, Joseph R. Mazzulli, et al. "Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2." Proceedings of the National Academy of Sciences 113, no. 14 (March 21, 2016): 3791–96. http://dx.doi.org/10.1073/pnas.1514005113.
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The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.
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Quraishi, Imran H., Anna M. Szekely, Anushree C. Shirali, Pramod K. Mistry, and Lawrence J. Hirsch. "Miglustat Therapy for SCARB2-Associated Action Myoclonus–Renal Failure Syndrome." Neurology Genetics 7, no. 5 (July 28, 2021): e614. http://dx.doi.org/10.1212/nxg.0000000000000614.
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ObjectiveWe evaluated whether substrate reduction therapy with miglustat could alter the course of action myoclonus–renal failure syndrome (AMRF), a rare, progressive myoclonic epilepsy with early mortality caused by scavenger receptor class B member 2 (SCARB2) gene mutations.MethodsWe identified an AMRF patient with a biallelic combination of SCARB2 mutations determined by whole exome sequencing. SCARB2 encodes a protein that traffics β-glucocerebrosidase to the lysosomal membrane. Mutations lead to a complex pattern of glucosylceramide accumulation and neurologic symptoms including progressive action myoclonus, seizures, and ataxia. We then evaluated the effect of inhibiting glucosylceramide synthesis, as is used in Gaucher disease. The patient was treated for 3 years with miglustat after several years of steady worsening.ResultsProgression of myoclonus halted, dysphagia resolved, some skills were reacquired, and seizures remained well controlled.ConclusionsThe response suggests that neurologic symptoms of SCARB2-associated AMRF could be ameliorated, at least partly, by targeting glycosphingolipid metabolism with available medications.
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Hodaňová, Kateřina, Martin Hřebı́ček, Markéta Červenková, Lenka Mrázová, Lenka Vepřeková, and Jiřı́ Zeman. "Analysis of the β-Glucocerebrosidase Gene in Czech and Slovak Gaucher Patients: Mutation Profile and Description of Six Novel Mutant Alleles." Blood Cells, Molecules, and Diseases 25, no. 5 (October 1999): 287–98. http://dx.doi.org/10.1006/bcmd.1999.0256.
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Srikanth, Manasa P., and Ricardo A. Feldman. "Elevated Dkk1 Mediates Downregulation of the Canonical Wnt Pathway and Lysosomal Loss in an iPSC Model of Neuronopathic Gaucher Disease." Biomolecules 10, no. 12 (December 3, 2020): 1630. http://dx.doi.org/10.3390/biom10121630.
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Gaucher Disease (GD), which is the most common lysosomal storage disorder, is caused by bi-allelic mutations in GBA1—a gene that encodes the lysosomal hydrolase β-glucocerebrosidase (GCase). The neuronopathic forms of GD (nGD) are characterized by severe neurological abnormalities that arise during gestation or early in infancy. Using GD-induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs), we have previously reported that neuronal cells have neurodevelopmental defects associated with the downregulation of canonical Wnt signaling. In this study, we report that GD NPCs display elevated levels of Dkk1, which is a secreted Wnt antagonist that prevents receptor activation. Dkk1 upregulation in mutant NPCs resulted in an increased degradation of β-catenin, and there was a concomitant reduction in lysosomal numbers. Consistent with these results, incubation of the mutant NPCs with recombinant Wnt3a (rWnt3a) was able to outcompete the excess Dkk1, increasing β-catenin levels and rescuing lysosomal numbers. Furthermore, the incubation of WT NPCs with recombinant Dkk1 (rDkk1) phenocopied the mutant phenotype, recapitulating the decrease in β-catenin levels and lysosomal depletion seen in nGD NPCs. This study provides evidence that downregulation of the Wnt/β-catenin pathway in nGD neuronal cells involves the upregulation of Dkk1. As Dkk1 is an extracellular Wnt antagonist, our results suggest that the deleterious effects of Wnt/β-catenin downregulation in nGD may be ameliorated by the prevention of Dkk1 binding to the Wnt co-receptor LRP6, pointing to Dkk1 as a potential therapeutic target for GBA1-associated neurodegeneration.
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Brown, Annie, Jiayi Zhang, Brendan Lawler, and Biao Lu. "Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1." Genes 12, no. 7 (July 15, 2021): 1076. http://dx.doi.org/10.3390/genes12071076.
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Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.
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Barese, Cecilia, Michael Waring, Richard Pfeifer, and Chris Mason. "Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients." Blood 132, Supplement 1 (November 29, 2018): 5791. http://dx.doi.org/10.1182/blood-2018-99-119766.
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Abstract The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system. We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting. Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens. Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells. Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method. Disclosures No relevant conflicts of interest to declare.
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Burbulla, Lena F., Sohee Jeon, Jianbin Zheng, Pingping Song, Richard B. Silverman, and Dimitri Krainc. "A modulator of wild-type glucocerebrosidase improves pathogenic phenotypes in dopaminergic neuronal models of Parkinson’s disease." Science Translational Medicine 11, no. 514 (October 16, 2019): eaau6870. http://dx.doi.org/10.1126/scitranslmed.aau6870.
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Mutations in the GBA1 gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase) represent the most common risk factor for Parkinson’s disease (PD). GCase has been identified as a potential therapeutic target for PD and current efforts are focused on chemical chaperones to translocate mutant GCase into lysosomes. However, for several GBA1-linked forms of PD and PD associated with mutations in LRRK2, DJ-1, and PARKIN, activating wild-type GCase represents an alternative approach. We developed a new small-molecule modulator of GCase called S-181 that increased wild-type GCase activity in iPSC-derived dopaminergic neurons from sporadic PD patients, as well as patients carrying the 84GG mutation in GBA1, or mutations in LRRK2, DJ-1, or PARKIN who had decreased GCase activity. S-181 treatment of these PD iPSC-derived dopaminergic neurons partially restored lysosomal function and lowered accumulation of oxidized dopamine, glucosylceramide and α-synuclein. Moreover, S-181 treatment of mice heterozygous for the D409V GBA1 mutation (Gba1D409V/+) resulted in activation of wild-type GCase and consequent reduction of GCase lipid substrates and α-synuclein in mouse brain tissue. Our findings point to activation of wild-type GCase by small-molecule modulators as a potential therapeutic approach for treating familial and sporadic forms of PD that exhibit decreased GCase activity.
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Gegg, Matthew E., Guglielmo Verona, and Anthony H. V. Schapira. "Glucocerebrosidase deficiency promotes release of α-synuclein fibrils from cultured neurons." Human Molecular Genetics 29, no. 10 (May 11, 2020): 1716–28. http://dx.doi.org/10.1093/hmg/ddaa085.
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Abstract Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most important genetic risk factor for Parkinson disease (PD). GCase activity is also decreased in sporadic PD brains and with normal ageing. Loss of GCase activity impairs the autophagy lysosomal pathway resulting in increased α-synuclein (α-syn) levels. Furthermore, elevated α-syn results in decreased GCase activity. Although the role of α-syn in PD remains unclear, evidence indicates that aggregated α-syn fibrils are a pathogenic species in PD, passing between neurons and inducing endogenous native α-syn to aggregate; spreading pathology through the brain. We have investigated if preformed α-syn fibrils (PFFs) impair GCase activity in mouse cortical neurons and differentiated dopaminergic cells, and whether GCase deficiency in these models increased the transfer of α-syn pathology to naïve cells. Neurons treated with PFFs induced endogenous α-syn to become insoluble and phosphorylated at Ser129 to a greater extent than monomeric α-syn-treatment. PFFs, but not monomeric α-syn, inhibited lysosomal GCase activity in these cells and induced the unfolded protein response. Neurons in which GCase was inhibited by conduritol β-epoxide did not increase the amount of insoluble monomeric α-syn or its phosphorylation status. Instead the release of α-syn fibrils from GCase deficient cells was significantly increased. Co-culture studies showed that the transfer of α-syn pathology to naïve cells was greater from GCase deficient cells. This study suggests that GCase deficiency increases the spread of α-syn pathology and likely contributes to the earlier age of onset and increased cognitive decline associated with GBA-PD.
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Serfecz, Jacquelyn C., Afsoon Saadin, Clayton P. Santiago, Yuji Zhang, Søren M. Bentzen, Stefanie N. Vogel, and Ricardo A. Feldman. "C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages." International Journal of Molecular Sciences 22, no. 18 (September 14, 2021): 9912. http://dx.doi.org/10.3390/ijms22189912.
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Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8–10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.
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Ortega-Rosales, Alberto, Carlos Burneo-Rosales, Gilda Romero-Ulloa, and Gabriela Burneo-Rosales. "Case Report: Pancytopenia as an indicator for lysosomal storage disease (Gaucher's Disease)." F1000Research 8 (October 10, 2019): 755. http://dx.doi.org/10.12688/f1000research.18802.2.
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Introduction: Lysosomal storage disorders are a rare group of diseases with genetic origin in which Gaucher Disease (GD) stands out as the most frequent. GD type 1 is the most common form of this condition, and patients with this pathology present with unexplained cytopenias, in addition to hepatosplenomegaly, bone involvement, and in other cases neurological disorders. A case of a patient is presented, whose results showed thrombocytopenia and leukopenia in addition to hepatosplenomegaly. In Ecuador, there are very few reported cases of this clinical entity, and information on this disease is very limited. Case: We present a case of a patient diagnosed with GD, who presented with thrombocytopenia and leukopenia in addition to hepatosplenomegaly, with the aim of emphasizing the importance of early recognition of this pathology, especially in patients with unexplained cytopenia’s or hepatosplenomegaly’s. In suspicion of GD, enzymatic quantification of β-glucocerebrosidase was performed, showing its deficit in addition to alteration in the GBA gene. Conclusion: We believe that a high index of suspicion together with enzymatic and genetic testing are essential for an early specific diagnosis, which will allow the administration of timely treatment and avoid irreversible complications in order to reduce morbidity and improve the clinical aspects of the patient.
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Ibraheem, Mohammad Fadhil, and Shaymaa Jamal Ahmed. "Clinical and Genetic Varieties of Gaucher Disease in Iraqi Children." Journal of Child Science 10, no. 01 (January 2020): e202-e206. http://dx.doi.org/10.1055/s-0040-1720956.
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AbstractGaucher disease (GD), which is due to a deficiency in the lysosomal enzyme β-glucocerebrosidase, is a rare genetic disorder. It is characterized by a wide variety of clinical manifestations and severity of symptoms, making it difficult to manage. A cross-sectional hospital-based genetic study was undertaken with 32 pediatric patients. We recruited 21 males and 11 females diagnosed with GD, with a male-to-female ratio of 1.91:1. The mean age of the study population was 8.79 ± 4.37 years with an age range from 8 months to 17 years. We included patients on clinical evaluation from 2011 to 2019. An enzyme assay test was used to measure β-glucosidase enzyme activity in leukocytes and the GBA gene study was performed by polymerase chain reaction technique. We found GD type 1 in 27 (84.37%) participants, GD type 3 in five (15.63%) participants, while none classified as GD type 2. The dominant mutation in GD 1 was N370S in 81.5%, of which two-thirds were homozygous. The second common mutation in this type of disease (L444P) was present in nine cases (40.9%), two of whom were homozygous (9.9%). Meanwhile, R463C was present in six cases (27.27%), of whom one was homozygous. In GD 3, the dominant mutation was L444P as seen in 80% of the patients followed by N370S and R463C in 20%. This study shows that the most common mutant allele in this study was N370S, followed by L444P. Further large-scale studies with more advanced designs are recommended to explore the sequences of GBA genes.
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Comper, Fabrizio, I.-Mei Yu, Petya Kalcheva, Carlos J. Miranda, Elisa Chisari, Clement Cocita, Samantha Correia, et al. "Generation of β-Glucocerebrosidase variants with increased half-life in human plasma for liver directed AAV gene therapy aimed at the treatment of Gaucher disease type 1." Molecular Genetics and Metabolism 132, no. 2 (February 2021): S27—S28. http://dx.doi.org/10.1016/j.ymgme.2020.12.047.
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Wei, Jianshe, Yoshiki Takamatsu, Ryoko Wada, Masayo Fujita, Gilbert Ho, Eliezer Masliah, and Makoto Hashimoto. "Therapeutic Potential of αS Evolvability for Neuropathic Gaucher Disease." Biomolecules 11, no. 2 (February 15, 2021): 289. http://dx.doi.org/10.3390/biom11020289.
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Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by autosomal recessive mutations of the glucocerebrosidase gene, GBA1. In the majority of cases, GD has a non-neuropathic chronic form with adult onset (GD1), while other cases are more acute and severer neuropathic forms with early onset (GD2/3). Currently, no radical therapies are established for GD2/3. Notably, GD1, but not GD2/3, is associated with increased risk of Parkinson’s disease (PD), the elucidation of which might provide a clue for novel therapeutic strategies. In this context, the objective of the present study is to discuss that the evolvability of α-synuclein (αS) might be differentially involved in GD subtypes. Hypothetically, aging-associated PD features with accumulation of αS, and the autophagy-lysosomal dysfunction might be an antagonistic pleiotropy phenomenon derived from αS evolvability in the development in GD1, without which neuropathies like GD2/3 might be manifested due to the autophagy-lysosomal dysfunction. Supposing that the increased severity of GD2/3 might be attributed to the decreased activity of αS evolvability, suppressing the expression of β-synuclein (βS), a potential buffer against αS evolvability, might be therapeutically efficient. Of interest, a similar view might be applicable to Niemann-Pick type C (NPC), another LSD, given that the adult type of NPC, which is comorbid with Alzheimer’s disease, exhibits milder medical symptoms compared with those of infantile NPC. Thus, it is predicted that the evolvability of amyloid β and tau, might be beneficial for the adult type of NPC. Collectively, a better understanding of amyloidogenic evolvability in the pathogenesis of LSD may inform rational therapy development.
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Ortega-Rosales, Alberto, Carlos Burneo-Rosales, Gilda Romero-Ulloa, and Gabriela Burneo-Rosales. "Case Report: Pancytopenia as an indicator for lysosomal storage disease (Gaucher's Disease)." F1000Research 8 (May 29, 2019): 755. http://dx.doi.org/10.12688/f1000research.18802.1.
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Introduction: Lysosomal storage disorders are a rare group of diseases with genetic origin in which Gaucher Disease (GD) stands out as the most frequent. GD type 1 is the most common form of this condition, and patients with this pathology present with unexplained cytopenias, in addition to hepatosplenomegaly, bone involvement, and in other cases neurological disorders. A case of a patient is presented, whose results showed thrombocytopenia and leukopenia in addition to hepatosplenomegaly. In Latin America, there are very few reported cases of this clinical entity, and information on this disease is very limited. Case: We present a case of a patient diagnosed with GD, who presented with thrombocytopenia and leukopenia in addition to hepatosplenomegaly, with the aim of emphasizing the importance of early recognition of this pathology, especially in patients with unexplained cytopenia’s or hepatosplenomegaly’s. In suspicion of GD, enzymatic quantification of β-glucocerebrosidase was performed, showing its deficit in addition to alteration in the GBA gene. Unfortunately, enzymatic replacement could not be done because the Cerazyme (imiglucerase for injection) is not available in Ecuador. Nevertheless, the patient was treated with analgesic (1g of paracetamol generally three times a day) and vitamin supplements (Dayamineral). Currently the patient is waiting for transfer to a foreign institution; she continues with bicytopenia and hepatosplenomegaly, her conditions are expected to be remit once the enzymatic treatment has been administered. Conclusion: We believe that the timely recognition of this disease will allow the initiation of enzymatic replacement therapy in an effective manner, in order to reduce morbidity and improve the clinical aspects of the patient.
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Giuffrida, Gaetano, Concetta Conticello, Daniela Nicolosi, Valeria Calafiore, Alessandra Romano, Ugo Consoli, Caterina Musolino, et al. "Preliminary Results of a Prospective Observational Study to Assess the Prevalence of Gaucher Disease in an Adult Population Affected By MGUS." Blood 134, Supplement_1 (November 13, 2019): 4868. http://dx.doi.org/10.1182/blood-2019-129944.
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INTRODUCTION: Gaucher disease (GD) is a rare, autosomal recessive genetic disorder. It is due to a deficiency of the lysosomal enzyme, glucocerebrosidase, which leads to an accumulation of its substrate, glucosylceramide, in tissue macrophage with damage to hematological, visceral, and skeletal organ systems. Although GD has a continuous spectrum of severity, it is traditionally classified into three forms: type 1 (chronic; lacking early onset neuronopathy), type 2 (acute; with early onset neuronopathy),and type 3 (chronic; with early onsetneuronopathy). Type 1GD accounts for more than 90% all GD patients. Its prevalence world-wide is 1 in 50,000-100,000 but it is as high as ~1 in 850 in individuals of Ashkenazi heritage. Type 1 GD is frequently associated to monoclonal gammopathies; despite the emergence of theories advanced to explain these observations, the cause remains unknown. OBJECTIVE: Aim of the ongoing observational study is to determine the prevalence of unrecognized type I GD in a selected Italian population with MGUS. MATERIALS AND METHODS: From January 2018, dried blood spots (DBS) sample from patients with laboratory evidence of MGUS coming from five hematology units of Sicily and Calabria were collected and tested for the acid β-glucosidase enzyme activity. The study was approved by the local institutional review board. All patients provided informed consent for the prospective collection of their data. In case of DBS positive result, a confirmatory test was carried over and, if GD was confirmed, the patient was referred to one of the Regional Reference Centers for Metabolic Disease, as for current clinical practice in Italy. RESULTS: To date, 308 patients with MGUS were enrolled; acid β-glucosidase enzyme activity was low in 22 patients (7%). Sequence analysis of GBA gene was performed in these selected patients, but we have found only 4 patients with heterozygous mutation in the GBA1 gene, 1 homozygous(c.1226A>G -N370S) and 1 compound heterozygous (c.1226A>G -N370S and c.1448T>C -L444P); the last 2 patients had signs of GD (hepato-splenomegaly and mild thrombocytopenia). CONCLUSIONS: Type 1 GD remains a rare lysosomal storage disorder but preliminary results of our observational study show that it should be considered in the diagnostic framework of patients with MGUS, particularly when other GD symptoms are present. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Di Raimondo:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding.
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Miranda, Carlos J., Miriam Canavese, Elisa Chisari, Jalpa Pandya, Clement Cocita, Maria Portillo, Jenny McIntosh, et al. "Liver-Directed AAV Gene Therapy for Gaucher Disease." Blood 134, Supplement_1 (November 13, 2019): 3354. http://dx.doi.org/10.1182/blood-2019-124280.
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Introduction: Gaucher disease (GD), one of the most common lysosomal storage disorders, is an autosomal recessive condition resulting from mutations in the GBA gene that codes for the b-glucocerebrosidase (GCase) enzyme. Over 90% of patients have type 1 GD, which is characterised by lipid engorged macrophages (known as Gaucher cells) in multiple organs, including spleen, liver and bone marrow, with no overt involvement of the central nervous system (CNS). The current standard of care for type 1 GD patients includes enzyme replacement therapy (ERT), which provides good overall therapeutic benefit. However, ERT is administered intravenously every other week, resulting in a high cumulative cost and a significant treatment burden. Furthermore, disease manifestations, such as pulmonary and skeletal disease, remain unresolved with ERT. Gene therapy is emerging as a very promising avenue of treatment for various monogenic disorders and has the potential to provide sustained levels of GCase enzyme expression after a single treatment. Here we have evaluated liver-directed gene therapy in vitro and in vivo for the treatment of GD. Methods: Adeno-associated virus (AAV) constructs were optimised to express full-length wild-type GCase protein (GBA AAV) and packaged in AAV8 capsids for in vivo mouse studies, or our novel AAVS3 capsid for in vitro studies in a human cell line. GCase activity was determined fluorometrically with 4-Methylumbelliferyl-β-D-glucopyranoside and activity was based on a 4-methylumbelliferone standard curve. Levels of GCase in plasma, and uptake in GD target organs were compared between our GBA AAV optimized construct and ERT treatment with velaglucerase alfa (VPRIV®) in C57BL/6 wild type mice. Doses used ranged from 2x109 to 2x1012 vg/kg for GBA AAV constructs and 60 U/kg for ERT. Results: Our initial proof of concept studies for liver-directed AAV gene therapy of GD used an AAV construct encoding the native full-length human GBA cDNA (RC-04-01). After a single intravenous injection into mice, RC-04-01 led to a dose-dependent expression of GCase in liver and robust levels of enzymatically active GCase in plasma. Based on these preliminary data, 37 GBA AAV constructs with optimisations to the coding sequence and changes to the promoter, signal peptide, and polyA sequences were designed and evaluated in vitro for GCase production. Among them, 6 constructs outperformed RC-04-01 and were further tested in mice. Construct RC-04-26 showed the highest GCase activity levels both in vivo and in vitro. At a dose of 2x1012 vg/kg, RC-04-26 showed plasma active GCase levels up to 9.4-fold higher than RC-04-01. Upon GBA AAV infusion, plasma GCase levels were steady and sustained for the duration of the entire study period (9 months). In addition, RC-04-26 infusion into mice resulted in robust uptake of GCase by cells in spleen, bone marrow and lung, demonstrating that liver-produced GCase is taken up by macrophages present in the GD target organs. Lastly, GCase bioavailability was evaluated after a single administration of RC-04-26 or ERT. With velaglucerase alfa, GCase was rapidly cleared from the bloodstream and tissues. However, RC-04-26 resulted in sustained and steady levels of GCase, with an overall bioavailability of GCase uptake over 170-fold higher than with ERT. Conclusions: Our current data support the hypothesis that a single administration of an optimised liver directed GBA AAV vector results in sustained elevation of GCase in the bloodstream and higher level of GCase bioavailability for uptake into macrophages than velaglucerase alfa. This observation supports further development of AAV gene therapy for Gaucher disease with the potential for enhanced therapeutic benefit from a one-off administration. Disclosures Miranda: Freeline Therapeutics: Employment, Equity Ownership. Canavese:Freeline Therapeutics: Employment, Equity Ownership. Chisari:Freeline Therapeutics: Employment, Equity Ownership. Pandya:Freeline: Employment, Equity Ownership. Cocita:Freeline Therapeutics: Employment, Equity Ownership. Portillo:Freeline: Employment, Equity Ownership. McIntosh:Freeline Therapeutics: Consultancy, Equity Ownership. Kia:Freeline Therapeutics: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Dane:Freeline: Employment, Equity Ownership. Jeyakumar:Freeline Therapeutics: Employment, Equity Ownership. Sheridan:Freeline Therapeutics: Employment, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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Delbini, Paola, Viola Ghiandai, Maria Domenica Cappellini, Lorena Duca, Isabella Nava, Elena Cassinerio, and Irene Motta. "Evaluation of in Vitro Macrophage Characterization in Gaucher Type 1 (GD1) Patients." Blood 134, Supplement_1 (November 13, 2019): 3592. http://dx.doi.org/10.1182/blood-2019-130032.
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Gaucher disease type 1 is the most common inherited lysosomal storage disorder caused by the deficiency of lysosomal β-glucocerebrosidase (GBA, acid-β-glucosidase), required for the degradation of glycosphingolipids. The deficiency of the enzyme results in the widespread accumulation of glucosylceramide in macrophages, leading to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). However, GD manifestations are caused not only by the burden of glucosylceramide storage, but also by macrophage activation. It seems that GD reflects the downstream consequences of inappropriate macrophage activation, with the release of pro-inflammatory cytokines and other responses to storage material. The aim of this study is to assess in vitro phenotypic characterization, functional properties and gene expression anaysis of GD1 macrophages in order to understand their possible role in inflammation and in impairment of iron metabolism occurring in GD patients. Monocytes were isolated from buffy coats of GD patients (n=3) and controls (n=3) by applying an in vitro model protocol. Monocytes were expanded in ImmunoCult™ medium and 1% ZellShield® antibiotic cocktail. Differentiation was induced in ImmunoCult™ medium, 50 ng/mL M-CSF and 50 ng/mL GM-CSF. To mimic the in vivo condition, macrophage population was loaded with erythrocytes ghosts isolated from GD patients. Cell morphology was analyzed on cytocentrifuged preparations stained with May-Grünwald Giemsa (MGG). Surface marker expression (CD11, CD33, CD68, CD64) were examined by flow cytometry to evaluate macrophages differentiation and phenotype. Gene expression analysis of iron metabolism-related genes was evaluated through Real-Time Quantitative PCR. Biochemical indices (NTBI, GDF15, sTfR and chitotriosidase) were analyzed in supernatant through ELISA assay. Flow cytometry analysis (Fig.1) revealed that without erythrocytes ghosts (preM∅), the proportion of CD11+/CD68+ macrophage population was similar between GD patients and control. However, in GD macrophages, when loaded with Gaucher erythrocytes ghosts (M∅+ghosts), the proportion of CD11++/CD68++ cells increased (36.4%), as a reflection of a more pro-inflammatory phenotype. Treated controls showed no differences. To further characterize these different macrophages subpopulations in GD patients, we used an additional parameter, CD64, because CD64/CD68 markers are specific for M1/M2 polarization. GD M∅+ghosts showed an higher percentage of CD64++/CD68++ population (78%) in comparison of GD pre-M∅ (45%) and controls (36%), confirming a M1 proinflammatory phenotype of GD macrophages loaded with erythrocytes ghosts. Under microscope evaluation, GD M∅+ghosts presented high number of spindle-shaped fibroblastoid, typically M1 phenotype cells, rather than large flat-round cells (M2 cells). Moreover, morphological staining of these cells confirmed the typical features of GD cells with basophilic cytoplasm with characteristic crinkles. Controls showed no differences in macrophage features when loaded with erythrocytes ghosts. To confirm the pro-inflammatory potential of GD M∅+ghosts, high levels of pro-inflammatory mediators TNF-α, IL-1β and (s)TfR) were found in supernatant of GD M∅+ghosts (72,9±8,5, 24,7±2,7 and 3,1±4,4, respectively) compared to pre-M∅ and controls. High GDF15 expression in GD M∅+ghosts (14,77±5,87) respect to preM∅ (1,80±1,1) and controls (1,67±0,8) was observed. By gene expression analysis we observed in GD M∅+ghosts an higher HAMP expression (28,13±5,63) compared to preM∅ (2,49±1,5) and controls (0,71±0,02), and a lower SLC40A1 expression (0,01±0,00) compared to GD preM∅ (0,16±0,05) and controls (0,79±0,25). No significant differences in TFRC and GDF11 expression between GD preM∅ and M∅+ghosts in GD patients were observed. These preliminary data suggest that GD macrophages, when stimulated, display a proinflammatory potential. These activated M1 macrophages could contribute to an inflammatory-producing environment, triggering hepcidin and ferroportin expression by an autocrine/paracrine mechanism and leading to dysregulation of iron distribution. Disclosures Cappellini: CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Ruchlemer, Rosa, Deborah Elstein, Eti Broide, Constantine Reinus, Hannah Maayaan, and Ari Zimran. "Gaucher Disease and LGL Proliferations: Provocative Commonality?" Blood 118, no. 21 (November 18, 2011): 1100. http://dx.doi.org/10.1182/blood.v118.21.1100.1100.
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Abstract Abstract 1100 Large granular lymphocytes(LGL)which may be of T or NK phenotype, normally account for 10–15% of peripheral blood(PB)lymphocytes. T-LGL leukemia is a clonal expansion of antigen-primed competent cytotoxic-T-lymphocytes(CTL)which is unique in that these cells retain many phenotypic and functional properties of normal cytotoxic effector T cells. Sphingolipid-mediated signaling has been shown to have a role in long-term survival of CTLs. The diagnosis of T-LGL leukemia requires proof of clonality, yet clonal T-LGL proliferations may occur in normal individuals with autoimmune diseases, viral infections, B cell lymphoproliferations and post bone marrow transplantation rendering the differentiation of malignant (leukemic) from benign LGL expansions at times difficult. It has been suggested that T-LGL proliferations with polyclonal or oligoclonal TCR gene rearrangement may be a response to a stimulus before the emergence of a dominant clone. Gaucher disease (GD)is a lysosomal storage disease caused by mutations in the gene encoding acid β-glucocerebrosidase, leading to the accumulation of glucocerebroside in tissue macrophages (Gaucher cells) and resulting in hepatosplenomegaly, cytopenias and skeletal involvement. GD and T-LGL leukemia share strikingly similar clinical features, including cytopenias, splenomegaly, B cell dyscrasias, arthralgias, pulmonary hypertension (PHT), and increased association with neoplasms and autoimmune disorders. The etiology of PHT, B cell dyscrasias, and the increased frequency of neoplasms in GD remains unclear. Table 1 describes the characteristics of 4 GD patients suffering from severe cytopenias relative to the overall phenotype of their GD in whom LGL were found to comprise the majority of the PB lymphocytes. Patient #1: 31year-old female suffering from severe GD with massive splenomegaly and severe cytopenias but refuses Gaucher-specific therapy; Patient #2: 31year-old female has suffered from severe Gaucher disease since childhood, was splenectomized, has unexplained anemia and known LGL expansion for 7 years but clonality studies continue to be negative; Patient #3: 45 year-old female with severe GD, splenectomized, suffered from AVN, bone crisis autoimmune hemolytic anemia, PHT, and B cell dyscrasia; and Patient #4: 72 year-old male has asymptomatic GD with progressive pancytopenia who was diagnosed with myelodysplastic syndrome(MDS),T-LGL proliferation and monclonal gammopathy of unknown origin(MGUS). LGL cells comprised 70% of PB lymphocytes in addition there was severe trilineage dysplastic changes, TCR rearrangement was positive. Interestingly, 8 months after the start of enzyme replacement therapy(ERT),counts improved and the number of PB LGL was reduced to <10% of lymphocytes. In a cohort of 55 consecutive GD patients, 38% had LGL expansions in the PB smears. Since both conditions may be asymptomatic for many years, their co-existence can easily be missed.Table 1:Patient characteristics1234Age at diagnosis31314572GenderFemaleFemaleFemaleMaleSplenectomyNoYesYesNoBony diseaseAVNBone crisis, AVN, osteomyelitisNo?Pulmonary HypertensionNoNoYesNoAutoimmune phenomenonNoNoAIHANoB cell dyscrasiasYesNoPolyclonal hypergammagloblulinemiaMGUSOther neoplasmsNoNoNoMDSLeucocyte count (×10/ul)2.213.36.62.9LGL percent of lymphocytes52%62%30%70%LGL absolute count (×10/ul)0.55.111.10.532LGL phenotypeCD3+,CD8+, CD56+, CD16−, CD57−, TCR αβ+CD3+, CD8+, CD16−, CD56−, TCR αβ+?CD3+, CD8+, CD16−, CD56−, TCR αβ+2 populationsa) CD3+, CD8+, CD56+, TCR αβ+9.3g/dlb) CD3−,CD56+,CD4−, CD8−Hemoglobin (g/dl)4.911.311.4Platelet count (x10/ul)111779630TCR rearrangementNegativeNegativePositivePositiveBone marrow pattern of involvementMild interstitial infiltrate of small lymphocytes CD8+TMild interstitial infiltrate of small lymphocytes CD8+TERTNoYesYesYesERT: improvement of cytopeniasUntreatedNoNoYes The co-existence of two hematological pathologies in more than one patient and the possibility that therapy for one may impact the other, is provocative. We suggest that LGL expansions in GD may be reactive to glucocereborside accumulation in Gaucher cells, and may play a pathogenic role in the development of some of the features of GD as well as induce/exacerbate some associated disorders. Disclosures: Zimran: Actelion: Honoraria.
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Arrant, Andrew E., Jonathan R. Roth, Nicholas R. Boyle, Shreya N. Kashyap, Madelyn Q. Hoffmann, Charles F. Murchison, Eliana Marisa Ramos, et al. "Impaired β-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations." Acta Neuropathologica Communications 7, no. 1 (December 2019). http://dx.doi.org/10.1186/s40478-019-0872-6.
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AbstractLoss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only β-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature β-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated β-glucocerebrosidase. Immunostaining revealed loss of neuronal β-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on β-glucocerebrosidase outside of the context of neurodegeneration, we investigated β-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn−/− mice had lower β-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs β-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired β-glucocerebrosidase processing.
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Brunialti, Electra, Alessandro Villa, Marianna Mekhaeil, Federica Mornata, Elisabetta Vegeto, Adriana Maggi, Donato A. Di Monte, and Paolo Ciana. "Inhibition of microglial β-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons." Journal of Neuroinflammation 18, no. 1 (September 22, 2021). http://dx.doi.org/10.1186/s12974-021-02272-2.
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Abstract Background Homozygotic mutations in the GBA gene cause Gaucher’s disease; moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinson’s disease. In homozygosis, these mutations impair the activity of β-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gaucher’s disease. Notwithstanding the key role of macrophages in this disease, most of the effects in the brain have been attributed to the β-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. Methods We applied the bioluminescence imaging technology, immunohistochemistry and gene expression analysis to investigate the consequences of microglial β-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia cocultures and in cell lines. The use of primary cells from reporter mice allowed for the first time, to discriminate in cocultures neuronal from microglial responses consequent to the β-glucocerebrosidase inhibition; results were finally confirmed by pharmacological depletion of microglia from the brain of mice. Results Our data demonstrate the existence of a novel neuroprotective mechanism mediated by a direct microglia-to-neuron contact supported by functional actin structures. This cellular contact stimulates the nuclear factor erythroid 2-related factor 2 activity in neurons, a key signal involved in drug detoxification, redox balance, metabolism, autophagy, lysosomal biogenesis, mitochondrial dysfunctions, and neuroinflammation. The central role played by microglia in this neuronal response in vivo was proven by depletion of the lineage in the brain of reporter mice. Pharmacological inhibition of microglial β-glucocerebrosidase was proven to induce morphological changes, to turn on an anti-inflammatory/repairing pathway, and to hinder the microglia ability to activate the nuclear factor erythroid 2-related factor 2 response, thus increasing the neuronal susceptibility to neurotoxins. Conclusion This mechanism provides a possible explanation for the increased risk of neurodegeneration observed in carriers of GBA mutations and suggest novel therapeutic strategies designed to revert the microglial phenotype associated with β-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication.
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Lepe-Balsalobre, Esperanza, José D. Santotoribio, Ramiro Nuñez-Vazquez, Salvador García-Morillo, Pilar Jiménez-Arriscado, Paula Hernández-Arévalo, Rocío Delarosa-Rodríguez, Juan M. Guerrero, and Hada C. Macher. "Genotype/phenotype relationship in Gaucher disease patients. Novel mutation in glucocerebrosidase gene." Clinical Chemistry and Laboratory Medicine (CCLM), June 25, 2020. http://dx.doi.org/10.1515/cclm-2020-0306.
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AbstractObjectivesGaucher disease (GD) is the most common inherited lysosomal storage disease, caused by mutations in acid β-glucosidase (GBA) gene. This study aimed to identify mutations in Andalusia patients with GD and their genotype-phenotype correlation.MethodsDescriptive observational study. University Hospital Virgen del Rocio patients diagnosed from GD from 1999 to 2019 were included. Demographic and clinical data, β-glucocerebrosidase activity, variants pathogenic in GBA gene and biomarkers for monitoring treatment were collected from digital medical record.ResultsTwenty-six patients with aged between 1 day and 52 years were studied. A total of six mutations described as pathogenic and one mutation not described above [c.937T>C (p.Tyr313His)] were identified in the GBA gene, four patients were homozygotes and 22 compound heterozygotes. Twenty-four patients were diagnosed in non-neuropathic form (type 1) and two cases presented neurological involvement (type 2 or 3). The most common variant was c.1226A>G (p.Asn409Ser), which was detected in 24 patients, followed by c.1448T>C (p.Leu483Pro) variant, identified in 13 patients. The c.1448T>C (p.Leu483Pro) mutation has been presented in the most severe phenotypes with neurological involvement associated with type 2 and 3 GD, while c.1226A>G (p.Asn409Ser) mutation has not been associated with neurological alterations. Splenomegaly and bone disease were the most frequent clinical manifestations, and thrombocytopenia was the most common hematological disorder.ConclusionsThe c.1226A>G (p.Asn409Ser) and c.1448T>C (p.Leu483Pro) mutations were the most common. The c.937T>C (p.Tyr313His) was identified as a novel mutation. The c.1448T>C (p.Leu483Pro) mutation was associated with neurological alterations and c.1226A>G (p.Asn409Ser) mutation has not been associated it.
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Lakočević, Milan B., Mirjana M. Platiša, Zorica R. Šumarac, Nada D. Suvajdžić, Lana Đ. Mačukanović, and Milan S. Petakov. "Peripheral neural response and sex hormones in type 1 gaucher disease." Journal of Medical Biochemistry, May 11, 2019. http://dx.doi.org/10.2478/jomb-2019-0020.
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Summary Background In a rare Gaucher disease, reduced activity of lysosomal β-glucocerebrosidase incompletely blocks glucosphingolipid catabolism. Accumulation of the unhydrolyzed substrate glucosylceramide within lysosomes results in progressive, multisystem Gaucher disease, classified into three types. Both parkinsonism and peripheral neuropathy are observed in cases of putative non-neuronopathic type 1 disease. In the current study we investigated whether the peripheral neural response in type 1 Gaucher disease patients, with no neural manifestations is conditioned by the influence of sex hormones. Methods The catalytic activity of β-glucocerebrosidase in peripheral blood leukocytes was determined spectrofluorometrically. Direct sequencing of the GBA1 gene was performed. Somatosensory evoked potentials were recorded after electrical stimulation of the median nerve of both arms. Stimuli of 0.2 ms duration at a frequency of 5 Hz were used. Sex hormones were determined by radioimmunoassay using a gamma scintillation counter. Results Analysis of the somatosensory evoked potentials revealed significant differences in peak latencies on periphery between men and women in both control and type 1 Gaucher disease groups. Analysis by gender showed significant associations between latencies and sex hormones only in female patients: negative correlation between oestradiol concentration and N9 peak latency, and a strong negative correlation of testosterone levels with all peak latencies on the periphery (N9-N13). Conclusions A relationship between testosterone concentrations and the latencies of potentials evoked on peripheral nerves exists only in females with type 1 Gaucher disease. We point out sexual dimorphism in the development of this entity.
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Brooker, Sarah M., and Dimitri Krainc. "Glucocerebrosidase dysfunction in neurodegenerative disease." Essays in Biochemistry, September 16, 2021. http://dx.doi.org/10.1042/ebc20210018.
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Abstract Parkinson’s disease (PD) and related neurodegenerative disorders, termed the synucleinopathies, are characterized pathologically by the accumulation of protein aggregates containing α-synuclein (aSyn), resulting in progressive neuronal loss. There is considerable need for the development of neuroprotective strategies to halt or slow disease progression in these disorders. To this end, evaluation of genetic mutations associated with the synucleinopathies has helped to elucidate crucial mechanisms of disease pathogenesis, revealing key roles for lysosomal and mitochondrial dysfunction. The GBA1 gene, which encodes the lysosomal hydrolase β-glucocerebrosidase (GCase) is the most common genetic risk factor for PD and is also linked to other neurodegenerative disorders including dementia with Lewy bodies (DLB). Additionally, homozygous mutations in GBA1 are associated with the rare lysosomal storage disorder, Gaucher’s disease (GD). In this review, we discuss the current knowledge in the field regarding the diverse roles of GCase in neurons and the multifactorial effects of loss of GCase enzymatic activity. Importantly, GCase has been shown to have a bidirectional relationship with aSyn, resulting in a pathogenic feedback loop that can lead to progressive aSyn accumulation. Alterations in GCase activity have furthermore been linked to multiple distinct pathways involved in neurodegeneration, and therefore GCase has emerged as a promising target for therapeutic drug development for PD and related neurodegenerative disorders, particularly DLB.
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Karaca, Emin, Sema Kalkan, Huseyin Onay, Ayca Aykut, Mahmut Coker, and Ferda Ozkinay. "Analysis of the β-glucocerebrosidase gene in Turkish Gaucher disease patients: mutation profile and description of a novel mutant allele." Journal of Pediatric Endocrinology and Metabolism 25, no. 9-10 (January 1, 2012). http://dx.doi.org/10.1515/jpem-2012-0155.
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Oftedal, Linn, Jodi Maple-Grødem, Marthe Gurine Gunnarsdatter Førland, Guido Alves, and Johannes Lange. "Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid." Scientific Reports 10, no. 1 (December 2020). http://dx.doi.org/10.1038/s41598-020-79104-5.
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AbstractLysosomal dysfunction is an emerging feature in the pathology of Parkinson’s disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl β-d-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze–thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at − 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze–thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.
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Huebecker, Mylene, Elizabeth B. Moloney, Aarnoud C. van der Spoel, David A. Priestman, Ole Isacson, Penelope J. Hallett, and Frances M. Platt. "Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson’s disease." Molecular Neurodegeneration 14, no. 1 (November 8, 2019). http://dx.doi.org/10.1186/s13024-019-0339-z.
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Abstract Background Haploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson’s disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD. Methods To study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD. Results The present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and β-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD. Conclusions These findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.
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García Sanz, Patricia. "Neuroinflammation in Parkinson´s Disease: role of cholesterol." Portal De Realidad Extendida de la Universidad de Sevilla, February 18, 2021. http://dx.doi.org/10.35466/ra2021n6390.
Full textAbstract:
Parkinson's disease (PD) is the most prevalent neurodegenerative movement disorders, where dopaminergic neurons in the Substantia Nigra (SNpc) are lost, resulting in a decrease in striatal dopamine and, subsequently, motor control failure and diagnostic symptoms of resting tremor, bradykinesia and muscle rigidity. Additionally, about 40% of PD patients display depression and anxiety, as a complex mental condition that worsens their prognosis and quality of life. These non-motor symptoms and others may appear at early phases (prodromal stages) as well as during the progression of the disease (advanced stages). Dopaminergic degeneration is associated with the appearance of Lewy bodies, which contain membrane structures and misfolded proteins such as α-synuclein (α-Syn). PD is a multifactorial pathology and the main risk factors are environmental factors, genetic susceptibility and age. The number of PD patients is increasing dramatically, and so is the corresponding social and economic burden. Nearly 85-90% of PD cases are sporadic, 10-15% are family cases and 5% have Mendelian inheritance. The central nervous system (CNS) can initiate an immune response against pathogens or endogenous danger signals, that is, it is immunocompetent. In addition, it can interact with the peripheral immune system, through the synthesis and release of neurotransmitters that can regulate the differentiation and functioning of both innate immunity cells and acquired immunity. In this way, the CNS can modulate the immune response and limit inflammation-induced tissue damage. Neuroinflammation is a complex integration of the responses of all cells present within the CNS, including the neurons, macroglia, microglia and the infiltrating leukocytes and is part of this innate immune response. It is initiated by the microglia, the macrophages of the CNS, which can be activated by various stimuli and in which astrocytes also participate. The entire inflammatory reaction must stop in order to maintain the structure and tissue homeostasis, including the removal of pathogens, dead cells, or other cellular debris, and the restoration of the tissue. If the insult persists or the mechanisms involved in terminating the inflammation are inadequate, chronic inflammation may arise. Furthermore, inflammation can also occur in response to molecules secreted by degenerating neurons, a condition called neuroinflammation, a crucial factor in neurodegenerative diseases. Neurodegenerative diseases are a heterogeneous group of CNS disorders whose etiology is still unknown. Although the molecular and cellular bases underlying these diseases are different, the final pathway in which several of these molecular or cellular events (oxidative stress, misfolded proteins, deficit of trophic factors, alteration of autophagy-lysosomal pathways, among others) in which they converge may be common (neuroinflammation), contributing over time to neuronal death through the activation of microglial populations in specific regions of brain The presence of gliosis is a relevant pathological feature in PD. In vivo imaging studies using PET show that PD patients have a significant increase in neuroinflammation markers in the bridge region, basal ganglia, striatum, and frontal and temporal cortex, compared to controls of the same age. While post-mortem immunohistological analyzes, in the brains of PD patients, reveal morphological changes in the microglia and overexpression of pro-inflammatory proteins such as HLA-DR, COX (cyclooxygenase) and iNOS (inducible nitric oxide synthase). These data have contributed to strengthen the idea that microglial activation can occur in the early stages of the disease. These cells can induce significant neurotoxic effects due to the excess production of cytotoxic factors such as interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and nitric oxide (NO). α-Syn may play a role in the microglial activation observed in SNpc and in the increase in the expression of MHC-II molecules. Mutations in the GBA1 gene are one of the main genetic risk factors for PD. This gene encodes an essential lysosomal enzyme called β-glucocerebrosidase (GCase), which breaks down the glycolipid glucocerebroside into glucose and ceramide. The reduced activity of GCase result in an increase in the amount of α-Syn, promoting its aggregation. These results have also been found in the brains of patients with sporadic PD. The concept of inflammarafts has recently been introduced, which are cholesterol-enriched lipid domains that are thought to act as platform mediating the cellular inflammatory response, possibly being controlled by cholesterol and sphingolipid metabolism and Apolipoprotein E. The ApoE / Cholesterol complex from astrocytes could bind to the TREM2 and TLR4 receptor on the inflamaraft surface of the microglia to trigger inflammation and phagocytosis. In the PD context, an alteration of the intracellular cholesterol content as occurs when a GCase deficiency occurs could induce a neuroinflammatory response in the microglia and astrocytes. These can be activated by the neurons' own oxidative stress or by added α-Syn secreted by these neurons, which can be transferred to the microglia and astrocytes, thus triggering the inflammatory response as well. Moreover, it has been seen that this alteration in cholesterol favors the α-Syn aggregation in lipid rafts, favoring its aggregation and altering the signaling through the TMR2 and TRL4 receptors. Therefore, it is suggested that, as a consequence of these mechanisms, GBA1 mutations ultimately increase interleukins and the NLRP3 inflammasome, activating the inflammatory response.