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1.
ACS Chem Neurosci ; 10(8): 3847-3858, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31264853

RESUMO

Heparan sulfate (HS) is a complex polysaccharide from the glycosaminoglycan (GAG) family that accumulates in tissues in several neurological lysosomal storage diseases known as mucopolysaccharidosis (MPS) disorders. The quantitation of HS in biological samples is important for studying MPS disorders but is very challenging because of its high molecular weight and heterogeneity. Recently, acid-catalyzed butanolysis followed by LC-MS/MS analysis has emerged as a promising method for the determination of HS. Butanolysis of HS produces fully desulfated disaccharide cleavage products which are detected by LC-MS/MS. Herein we describe the synthesis of butylated HS disaccharide standards and their use for determining the identity of major product peaks in LC-MS chromatograms from butanolysis of HS as well as the related GAGs heparin and heparosan. Furthermore, synthesis of a d9-labeled disaccharide internal standard enabled the development of a quantitative LC-MS/MS assay for HS. The assay was utilized for the analysis of MPS IIIA mouse brain tissues, revealing significant differences in abundance and in the regional accumulation of the various HS disaccharides in affected mice.

2.
Org Biomol Chem ; 16(45): 8791-8803, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30403218

RESUMO

The quantification of heparan sulfate (HS) in biological matrices, e.g., urine, cerebrospinal fluid, tissue samples etc., is of great importance for the diagnosis and prognosis of several of the mucopolysaccharidosis (MPS) disorders, which are lysosomal storage diseases of impaired glycosaminoglycan metabolism. The development of suitable assays for this purpose is challenging due to the high molecular weight and complexity of HS. Recent efforts towards this goal include the acid catalysed methanolysis of HS, which desulfates the polymer and results in the formation of disaccharide cleavage products which can be detected and quantified by LC-MS/MS. We have synthesized a library of 12 HS-derived disaccharides as methanolysis standards via the stereoselective 1,2-cis glycosylation of suitably protected GlcA and IdoA acceptors with a 2-deoxy-2-azido thioglucoside donor. This facilitated identification of the major peaks in the LC-MS/MS chromatograms, and potentially will allow the monitoring of specific metabolites as surrogate markers for genotype. This work also paves the way towards a fully quantitative LC-MS/MS assay for HS via the preparation of a suitably labelled derivative.

3.
Hum Mutat ; 39(12): 1788-1802, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30118150

RESUMO

Maroteaux-Lamy syndrome (MPS VI) is an autosomal recessive lysosomal storage disorder caused by pathogenic ARSB gene variants, commonly diagnosed through clinical findings and deficiency of the arylsulfatase B (ASB) enzyme. Detection of ARSB pathogenic variants can independently confirm diagnosis and render genetic counseling possible. In this review, we collect and summarize 908 alleles (201 distinct variants, including 3 polymorphisms previously considered as disease-causing variants) from 478 individuals diagnosed with MPS VI, identified from literature and public databases. Each variant is further analyzed for clinical classification according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results highlight the heterogeneity of ARSB alleles, with most unique variants (59.5%) identified as missense and 31.7% of unique alleles appearing once. Only 18% of distinct variants were previously recorded in public databases with supporting evidence and clinical significance. ACMG recommends publishing clinical and biochemical data that accurately characterize pathogenicity of new variants in association with reporting specific alleles. Variants analyzed were sent to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and MPS VI locus-specific database (http://mps6-database.org) where they will be available. High clinical suspicion coupled with diagnostic testing for deficient ASB activity and timely submission and classification of ARSB variants with biochemical and clinical data in public databases is essential for timely diagnosis of MPS VI.

4.
Biochim Biophys Acta Mol Cell Res ; 1865(10): 1451-1464, 2018 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-29981367

RESUMO

Heparan acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT) is a lysosomal multi-pass transmembrane protein whose deficiency may lead to an accumulation of heparan sulphate and the neurodegenerative lysosomal storage disorder mucopolysaccharidosis (MPS) IIIC. In this study, HGSNAT activity was detected in extracellular vesicles isolated from both human urine and culture medium conditioned with HEK 293T cells. We also demonstrate that HGSNAT co-immunoprecipitates with antibodies to ALIX, which is associated with the endosomal sorting complexes required for transport (ESCRT) proteins, and is implicated in the targeting of proteins to intraluminal vesicles of multivesicular bodies, the origin of exosomes. Furthermore, mutation of a putative LYPXnL-based binding site within HGSNAT for the V-domain of ALIX ablated association of HGSNAT with ALIX, post-translational maturation, and transport through the endo-lysosomal network. Unexpectedly, however, a mutation within the V-domain of ALIX demonstrated enhanced HGSNAT association, perhaps due to the actual involvement of other binding sites in this interaction. Indeed, HGSNAT still co-immunoprecipitates with truncations of ALIX lacking the V-domain. Interestingly, CRISPR/Cas9 mediated knock-down of ALIX did not inhibit HGSNAT trafficking through the endo-lysosomal network, suggesting that there is an alternative pathway for trafficking HGSNAT that does not require ALIX. Nonetheless, the targeting of HGSNAT to extracellular vesicles may provide a mechanism to subsequently transfer this enzyme extracellularly to provide a foundation for a therapy for MPS IIIC patients.

5.
Exp Neurol ; 303: 38-47, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29408731

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from the deficit of the N-sulfoglucosamine sulfohydrolase (SGSH) enzyme that leads to accumulation of partially-degraded heparan sulfate. MPS IIIA is characterized by severe neurological symptoms, clinically presenting as Sanfilippo syndrome, for which no effective therapy is available. The lysosomal SGSH enzyme is conserved in Drosophila and we have identified increased levels of heparan sulfate in flies with ubiquitous knockdown of SGSH/CG14291. Using neuronal specific knockdown of SGSH/CG14291 we have also observed a higher abundance of Lysotracker-positive puncta as well as increased expression of GFP tagged Ref(2)P supporting disruption to lysosomal function. We have also observed a progressive defect in climbing ability, a hallmark of neurological dysfunction. Genetic screens indicate proteins and pathways that can functionally modify the climbing phenotype, including autophagy-related proteins (Atg1 and Atg18), superoxide dismutase enzymes (Sod1 and Sod2) and heat shock protein (HSPA1). In addition, reducing heparan sulfate biosynthesis by knocking down sulfateless or slalom expression significantly worsens the phenotype; an important observation given that substrate inhibition is being evaluated clinically as a treatment for MPS IIIA. Identifying the cellular pathways that can modify MPS IIIA neuropathology is an essential step in the development of novel therapeutic approaches to prevent and/or ameliorate symptoms in children with Sanfilippo syndrome.

6.
Behav Brain Res ; 336: 177-181, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28887197

RESUMO

The recent development of knock-in mouse models of Alzheimer's disease provides distinct advantages over traditional transgenic mouse models that rely on over-expression of amyloid precursor protein. Two such knock-in models that have recently been widely adopted by Alzheimer's researchers are the AppNL-F and AppNL-G-F mice. This study aimed to further characterise the behavioural phenotype and amyloid plaque distribution of AppNL-G-F/NL-G-F (C57BL/6J background) mice at six-months of age. An attempt to replicate a previous study that observed deficits in working memory in the Y-maze, showed no difference between AppNL-G-F/NL-G-F and wild-type mice. Further assessment of these mice using the novel object recognition test and Morris water maze also revealed no differences between AppNL-G-F/NL-G-F and wild-type mice. Despite a lack of demonstrated cognitive deficits, we report a reduction in locomotor/exploratory activity in an open field. Histological examination of AppNL-G-F/NL-G-F mice showed widespread distribution of amyloid plaques at this age. We conclude that whilst at six-months of age, memory deficits are not sufficiently robust to be replicated in varying environments, amyloid plaque burden is significant in AppNL-G-F/NL-G-F knock-in brain.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Placa Amiloide/genética , Placa Amiloide/patologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Técnicas de Introdução de Genes , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/genética , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placa Amiloide/metabolismo
7.
Exp Neurol ; 295: 243-255, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28601604

RESUMO

Axonal dystrophy has been described as an early pathological feature of neurodegenerative disorders including Alzheimer's disease and amyotrophic lateral sclerosis. Axonal inclusions have also been reported to occur in several neurodegenerative lysosomal storage disorders including Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome). This disorder results from a mutation in the gene encoding the lysosomal sulphatase sulphamidase, and as a consequence heparan sulphate accumulates, accompanied by secondarily-stored gangliosides. The precise basis of symptom generation in MPS IIIA has not been elucidated, however axonal dystrophy may conceivably lead to impaired vesicular trafficking, neuronal dysfunction and/or death. We have utilised a faithful murine model of MPS IIIA to determine the spatio-temporal profile of neuronal inclusion formation and determine the effect of restoring normal lysosomal function. Dopaminergic (tyrosine hydroxylase-positive), cholinergic (choline acetyltransferase-positive) and GABAergic (glutamic acid decarboxylase65/67-positive) neurons were found to exhibit axonal dystrophy in MPS IIIA mouse brain. Axonal lesions present by ~seven weeks of age were Rab5-positive but lysosomal integral membrane protein-2 negative, suggesting early endosomal involvement. By 9-12-weeks of age, immunoreactivity for the autophagosome-related proteins LC3 and p62 and the proteasomal subunit 19S was noted in the spheroidal structures, together with wildtype α-synuclein, phosphorylated Thr-181 Tau and amyloid precursor protein, indicative of impaired axonal trafficking. Sulphamidase replacement reduced but did not abrogate the axonal lesions. Therefore, if axonal dystrophy impairs neuronal activity and ultimately, neuronal function, its incomplete resolution warrants further investigation.


Assuntos
Axônios/patologia , Encéfalo/patologia , Mucopolissacaridose III/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Feminino , Hidrolases/genética , Imuno-Histoquímica , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose III/diagnóstico por imagem , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
8.
Mol Genet Metab ; 121(3): 259-270, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28532689

RESUMO

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase activity that leads to accumulation of glucosylceramide and glucosylsphingosine. Membrane raft microdomains are discrete, highly organized microdomains with a unique lipid composition that provide the necessary environment for specific protein-lipid and protein-protein interactions to take place. In this study we purified detergent resistant membranes (DRM; membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. We observed significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine leading to an altered DRM composition. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt DRM architecture making them less ordered and more fluid. In addition, significant changes in the length and degree of lipid saturation within the DRM microdomains in the Gaucher brain were also observed. As these DRM microdomains are involved in many cellular events, an imbalance or disruption of the cell membrane homeostasis may impair normal cell function. This disruption of membrane raft microdomains and imbalance within the environment of cellular membranes of neuronal cells may be a key factor in initiating a cascade process leading to neurodegeneration.


Assuntos
Doença de Gaucher/metabolismo , Lipídeos/química , Microdomínios da Membrana/química , Baço/química , Animais , Encéfalo/patologia , Química Encefálica , Colesterol/análise , Galactosiltransferases/análise , Gangliosídeos/análise , Glucosilceramidas/análise , Fosfatidilcolinas/análise , Ovinos , Baço/patologia
9.
J Inherit Metab Dis ; 40(5): 715-724, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28451919

RESUMO

Mucopolysaccharidosis (MPS) type IIIA, or Sanfilippo syndrome, is a neurodegenerative lysosomal storage disorder caused by a deficiency of the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), involved in the catabolism of heparan sulfate. The clinical spectrum is broad and the age of symptom onset and the degree of preservation of cognitive and motor functions appears greatly influenced by genotype. To explore this further, we generated a conditional knockout (Sgsh KO ) mouse model with ubiquitous Sgsh deletion, and compared the clinical and pathological phenotype with that of the spontaneous Sgsh D31N MPS-IIIA mouse model. Phenotypic deficits were noted in Sgsh KO mice prior to Sgsh D31N mice, however these outcomes did not correlate with any shift in the time of appearance nor rate of accumulation of primary (heparan sulfate) or secondary substrates (GM2/GM3 gangliosides). Other disease lesions (elevations in lysosomal integral membrane protein-II expression, reactive astrocytosis and appearance of ubiquitin-positive inclusions) were also comparable between affected mouse strains. This suggests that gross substrate storage and these neuropathological markers are neither primary determinants, nor good biomarkers/indicators of symptom generation, confirming similar observations made recently in MPS-IIIA patients. The Sgsh KO mouse will be a useful tool for elucidation of the neurological basis of disease and assessment of the clinical efficacy of new treatments for Sanfilippo syndrome.


Assuntos
Hidrolases/metabolismo , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
10.
J Inherit Metab Dis ; 40(3): 443-453, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27832416

RESUMO

Intra-cerebrospinal fluid (CSF) injection of recombinant human lysosomal enzyme is a potential treatment strategy for several neurodegenerative lysosomal storage disorders including Sanfilippo syndrome (Mucopolysaccharidosis type IIIA; MPS IIIA). Here we have utilised the MPS IIIA Huntaway dog model to compare the effectiveness of the repeated intermittent bolus injection strategy being used in the trials with an alternate approach; slow, continual infusion of replacement enzyme (recombinant human sulphamidase; rhSGSH) into the spinal CSF using a SynchroMed II® pump attached to a spinal infusion cannula. The ability of each enzyme delivery strategy to ameliorate lesions in MPS IIIA brain was determined in animals treated from ∼three- to six-months of age. Controls received buffer or no treatment. Significant reductions in heparan sulphate (primary substrate) were observed in brain samples from dogs treated via either cisternal or lumbar spinal CSF bolus injection methods and also in slow intra-spinal CSF infusion-treated dogs. The extent of the reduction differed regionally. Pump-delivered rhSGSH was less effective in reducing secondary substrate (GM3 ganglioside) in deeper aspects of cerebral cortex, and although near-amelioration of microglial activation was seen in superficial (but not deep) layers of cerebral cortex in both bolus enzyme-treated groups, pump-infusion of rhSGSH had little impact on microgliosis. While continual low-dose infusion of rhSGSH into MPS IIIA dog CSF reduces disease-based lesions in brain, it was not as efficacious as repeated cisternal or spinal CSF bolus infusion of rhSGSH over the time-frame of these experiments.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Líquido Cefalorraquidiano/metabolismo , Hidrolases/administração & dosagem , Vértebras Lombares/metabolismo , Mucopolissacaridose III/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Proteínas Recombinantes/administração & dosagem , Animais , Modelos Animais de Doenças , Cães , Terapia de Reposição de Enzimas/métodos , Heparitina Sulfato/metabolismo , Humanos , Mucopolissacaridose III/metabolismo , Doenças Neurodegenerativas/metabolismo
11.
Biochim Biophys Acta Proteins Proteom ; 1865(3): 261-273, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28007442

RESUMO

Parkinson's disease (PD) presents with neuropathological inclusions called Lewy bodies, which are primarily composed of fibrillar α-synuclein. Recently, we characterized sheep with Gaucher disease and since GBA1 mutations represent the highest genetic risk factor for PD, we have investigated α-synuclein fibrillation in the sheep. Here we demonstrate that differences in six amino acid residues between sheep and human α-synuclein significantly alter in vitro fibril formation. Circular dichroism of recombinant human and sheep α-synuclein show that both proteins adopt the same secondary structure. Fibrils from human and sheep α-synuclein formed at pH7.0 or 4.5 were analyzed by Transmission Electron Microscopy (TEM). Unexpectedly, sheep α-synuclein form fibrils much less readily than human α-synuclein and this difference was more pronounced at the lysosomal pH of 4.5. Aggregation-propensity and intrinsic-solubility analysis revealed that sheep α-synuclein had lower aggregation-propensity and higher solubility. As a result of these observations, TEM was used to analyze fibrils formed at pH4.5 of various "sheep-like" human or "human-like" sheep mutant α-synucleins, together with their wild-type forms. Thioflavin T was used to monitor in situ α-synuclein fibril formation at pH7.0 and 4.5. Results show that "sheep-like" human α-synuclein has substantially lower fibril aggregation, and "human-like" sheep α-synuclein aggregates faster than wild-type forms, respectively. Seeding with WT human α-synuclein showed that "sheep-like" human α-synuclein could not be seeded, providing further evidence that sheep sequence is resistant to fibrillation. These findings provide new avenues to prevent/reduce fibrillation in PD, which may aid in the development of therapies.


Assuntos
Amiloide/metabolismo , Ovinos/metabolismo , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Amiloide/genética , Animais , Humanos , Concentração de Íons de Hidrogênio , Cinética , Corpos de Lewy/genética , Corpos de Lewy/metabolismo , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Alinhamento de Sequência , Ovinos/genética , Solubilidade , Tiazóis/metabolismo , alfa-Sinucleína/genética
12.
J Neurochem ; 140(5): 703-717, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28027395

RESUMO

Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-ß in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD.


Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Autofagia , Lisossomos/patologia , Doença de Alzheimer/metabolismo , Animais , Humanos , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/patologia , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/fisiopatologia , Lisossomos/metabolismo
13.
Biochem Biophys Res Commun ; 479(4): 721-727, 2016 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-27693584

RESUMO

Pompe disease is caused by a deficiency in the lysosomal enzyme α-glucosidase, and this leads to glycogen accumulation in the autolysosomes of patient cells. Glycogen storage material is exocytosed at a basal rate in cultured Pompe cells, with one study showing up to 80% is released under specific culture conditions. Critically, exocytosis induction may reduce glycogen storage in Pompe patients, providing the basis for a therapeutic strategy whereby stored glycogen is redirected to an extracellular location and subsequently degraded by circulating amylases. The focus of the current study was to identify compounds capable of inducing rapid glycogen exocytosis in cultured Pompe cells. Here, calcimycin, lysophosphatidylcholine and α-l-iduronidase each significantly increased glycogen exocytosis compared to vehicle-treated controls. The most effective compound, calcimycin, induced exocytosis through a Ca2+-dependent mechanism, although was unable to release a pool of vesicular glycogen larger than the calcimycin-induced exocytic pore. There was reduced glycogen release from Pompe compared to unaffected cells, primarily due to increased granule size in Pompe cells. Drug induced exocytosis therefore shows promise as a therapeutic approach for Pompe patients but strategies are required to enhance the release of large molecular weight glycogen granules.


Assuntos
Calcimicina/farmacologia , Exocitose/efeitos dos fármacos , Doença de Depósito de Glicogênio Tipo II/metabolismo , Glicogênio/metabolismo , Iduronidase/farmacologia , Lisofosfatidilcolinas/farmacologia , Calcimicina/uso terapêutico , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Humanos , Iduronidase/uso terapêutico , Lisofosfatidilcolinas/uso terapêutico , Lisossomos/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Veículos Farmacêuticos/farmacologia
14.
BMC Vet Res ; 12(1): 136, 2016 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-27370326

RESUMO

The release of new DNA-based diagnostic tools has increased tremendously in companion animals. Over 70 different DNA variants are now known for the cat, including DNA variants in disease-associated genes and genes causing aesthetically interesting traits. The impact genetic tests have on animal breeding and health management is significant because of the ability to control the breeding of domestic cats, especially breed cats. If used properly, genetic testing can prevent the production of diseased animals, causing the reduction of the frequency of the causal variant in the population, and, potentially, the eventual eradication of the disease. However, testing of some identified DNA variants may be unwarranted and cause undo strife within the cat breeding community and unnecessary reduction of gene pools and availability of breeding animals. Testing for mucopolysaccharidosis Type VI (MPS VI) in cats, specifically the genetic testing of the L476P (c.1427T>C) and the D520N (c.1558G>A) variants in arylsulfatase B (ARSB), has come under scrutiny. No health problems are associated with the D520N (c.1558G>A) variant, however, breeders that obtain positive results for this variant are speculating as to possible correlation with health concerns. Birman cats already have a markedly reduced gene pool and have a high frequency of the MPS VI D520N variant. Further reduction of the gene pool by eliminating cats that are heterozygous or homozygous for only the MPS VI D520N variant could lead to more inbreeding depression effects on the breed population. Herein is debated the genetic testing of the MPS VI D520N variant in cats. Surveys from different laboratories suggest the L476P (c.1427T>C) disease-associated variant should be monitored in the cat breed populations, particularly breeds with Siamese derivations and outcrosses. However, the D520N has no evidence of association with disease in cats and testing is not recommended in the absence of L476P genotyping. Selection against the D520N is not warranted in cat populations. More rigorous guidelines may be required to support the genetic testing of DNA variants in all animal species.


Assuntos
Doenças do Gato/genética , Testes Genéticos/veterinária , Variação Genética , Mucopolissacaridose VI/veterinária , Animais , Cruzamento , Doenças do Gato/prevenção & controle , Gatos , Testes Genéticos/ética , Testes Genéticos/normas , Genótipo , Mucopolissacaridose VI/genética
15.
Hum Gene Ther ; 27(5): 363-75, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26975339

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Hidrolases/genética , Mucopolissacaridose III/genética , Animais , Anticorpos Neutralizantes/imunologia , Autofagia , Biomarcadores , Encéfalo/metabolismo , Proteínas de Ligação a DNA , Dependovirus/classificação , Modelos Animais de Doenças , Endossomos/metabolismo , Ativação Enzimática , Feminino , Gangliosídeo G(M3)/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vetores Genéticos/normas , Proteína Glial Fibrilar Ácida/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de Grupo de Alta Mobilidade , Humanos , Hidrolases/imunologia , Hidrolases/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/terapia , Proteínas de Saccharomyces cerevisiae , Transdução Genética
16.
Neurobiol Dis ; 91: 143-54, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26976737

RESUMO

Gaucher disease arises from mutations in the ß-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the ß-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in ß-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.


Assuntos
Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Glicoesfingolipídeos/metabolismo , Lisossomos/metabolismo , Doença Aguda , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios/metabolismo , Fenótipo , Ovinos , Baço/metabolismo
17.
J Neurochem ; 137(3): 409-22, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26762778

RESUMO

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual infusion of enzyme using pumps. We observed significant reductions in substrate accumulation and other disease-based lesions in treated mouse brain. Thus, the strategy warrants consideration for testing in large animal models of MPS IIIA and also in other neurodegenerative lysosomal storage disorders.


Assuntos
Encéfalo/patologia , Terapia de Reposição de Enzimas/métodos , Hidrolases/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Animais , Biomarcadores/metabolismo , Química Encefálica , Gliose/tratamento farmacológico , Gliose/patologia , Heparitina Sulfato/metabolismo , Humanos , Hidrolases/administração & dosagem , Bombas de Infusão Implantáveis , Ventrículos Laterais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Medula Espinal/metabolismo
18.
Exp Neurol ; 278: 11-21, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26626972

RESUMO

AIM: To determine the capacity of continual low-dose lysosomal enzyme infusion into the cerebrospinal fluid of mucopolysaccharidosis type IIIA (MPS IIIA) mice to reverse established neurodegenerative disease. The rationale behind the study is that there is only limited animal model-derived evidence supporting treatment of symptomatic patients, principally because few studies have been designed to examine disease reversibility. METHODS: Twelve-week old MPS IIIA mice were implanted with indwelling unilateral intra-ventricular cannulae. These were connected to subcutaneous mini-osmotic pumps infusing recombinant human sulphamidase. Pump replacement was carried out in some mice at 16-weeks of age, enabling treatment to continue for a further month. Control affected/unaffected mice received vehicle via the same method. Behavioural, neuropathological and biochemical parameters of disease were assessed. RESULTS: Improvement in some, but not all, behavioural parameters occurred. Sulphamidase infusion mediated a statistically significant reduction in primary (heparan sulphate) and secondary (gangliosides GM2, GM3) substrate accumulation in the brain, with small reductions in micro- but not astro-gliosis. There was no change in axonal spheroid number. All mice developed a humoural response, however the antibodies were non-neutralising and no adverse clinical effects were observed. CONCLUSIONS: Continual infusion of replacement enzyme partially ameliorates clinical, histological and biochemical aspects of MPS IIIA mice, when treatment begins at an early symptomatic stage.


Assuntos
Encéfalo/metabolismo , Hidrolases/administração & dosagem , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Animais , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Sistemas de Liberação de Medicamentos , Feminino , Gangliosídeo G(M3)/metabolismo , Gangliosidoses GM2/metabolismo , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Mucopolissacaridose III/complicações , Doenças Neurodegenerativas/etiologia , Sulfatases/genética , Sulfatases/metabolismo
19.
JIMD Rep ; 29: 59-68, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26620043

RESUMO

Mucopolysaccharidosis IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of lysosomal sulfamidase results in accumulation of heparan sulfate and secondary storage of glycolipids in the brain. Intra-cisternal sulfamidase infusions reduce disease-related neuropathology; however, repeated injections may subject patients to the risk of infection and tissue damage so alternative approaches are required. We undertook a proof-of-principle study comparing the ability of slow/continual or repeat/bolus infusion to ameliorate neuropathology in MPS IIIA mouse brain. Six-week-old MPS IIIA mice were implanted with subcutaneously located mini-osmotic pumps filled with recombinant human sulfamidase (rhSGSH) or vehicle, connected to lateral ventricle-directed cannulae. Pumps were replaced at 8 weeks of age. Additional MPS IIIA mice received intra-cisternal bolus infusions of the same amount of rhSGSH (or vehicle), at 6 and 8 weeks of age. Unaffected mice received vehicle via each strategy. All mice were euthanised at 10 weeks of age and the brain was harvested to assess the effect of treatment on neuropathology. Mice receiving pump-delivered rhSGSH exhibited highly significant reductions in lysosomal storage markers (lysosomal integral membrane protein-2, GM3 ganglioside and filipin-positive lipids) and neuroinflammation (isolectin B4-positive microglia, glial fibrillary acidic protein-positive astroglia). MPS IIIA mice receiving rhSGSH via bolus infusion displayed reductions in these markers, but the effectiveness of the strategy was inferior to that seen with slow/pump-based delivery. Continual low-dose infusion may therefore be a more effective strategy for enzyme delivery in MPS IIIA.

20.
Orphanet J Rare Dis ; 10: 143, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26537923

RESUMO

BACKGROUND: Fucosidosis results from lack of α-L-fucosidase activity, with accumulation of fucose-linked substrates in the nervous system and viscera leading to progressive motor and mental deterioration, and death. The naturally occurring dog model of fucosidosis was used to evaluate the neuropathological responses to partial enzyme replacement, and substrate reduction in early disease following treatment with recombinant canine α-L-fucosidase delivered through cerebrospinal fluid. METHODS: Neuropathology in both treated (n = 3) and untreated fucosidosis-affected (n = 3) animals was evaluated with immunohistochemistry, image analysis, manual quantification and gene expression analysis and compared with unaffected age-matched controls (n = 3) in an extension of our previous biochemical report on the same cohort. Data were analyzed by ANOVA. RESULTS: Quantification demonstrated a consistent trend to reduction in vacuolation, pyramidal neuron loss, astrocytosis, microgliosis, perivascular storage, apoptosis, oligodendrocyte loss, and hypomyelination throughout the central nervous system of enzyme treated animals compared to placebo-treated, age-matched affected controls. Key lesions including lysosomal expansion in neurons of deep cortex, astrocytosis in cerebral cortex and medulla, and increased lysosomal membrane associated protein-1 (LAMP-1) gene expression were ameliorated in treated animals. There was no change in spheroid formation and loss of Purkinje cells, but Purkinje cell vulnerability to apoptosis was reduced with treatment. CONCLUSIONS: Despite reduced severity of fucosidosis neuropathology with partial enzyme replacement, more complete and sustained biochemical correction is required to halt neuropathological processes in this large animal model of lysosomal storage disease.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Fucosidose/tratamento farmacológico , Fucosidose/patologia , alfa-L-Fucosidase/administração & dosagem , Animais , Cisterna Magna , Cães , Infusão Espinal , Resultado do Tratamento
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