Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2.

Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.

Essential role of excessive tryptophan and its neurometabolites in fatigue.

PURPOSE: Serotonin, a neurotransmitter synthesized from tryptophan, has been proposed to play a key role in central fatigue. In this study, we examined whether tryptophan itself and/or its two metabolites, kyneurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue. MATERIALS AND METHODS: Experiments were conducted using Sprague-Dawley rats (SDR) and Nagase analbuminemic rats (NAR). Central fatigue was assessed by treadmill running and a Morris water maze test. Microdialysis was used to collect samples for measurement of extracellular concentration of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) and to infuse test agents. To examine the kinetics of release, synaptosomes in the striatum were prepared in vitro to measure intra- and extrasynaptosomal concentration of tryptophan, serotonin and 5-HIAA. RESULTS: The concentration of tryptophan secreted into the extracellular space of the striatum was higher during fatigue only, and quickly returned to basal levels with recovery from fatigue. Running time to exhaustion was reduced by activation of tryptophan receptors. Time to exhaustion was shorter in NAR, which maintain a higher extracellular level of striatum tryptophan than SDR. Impaired memory performance in a water maze task after tryptophan treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. When branched-chain amino acids were administered, tryptophan transport to the extracellular space of the striatum was drastically inhibited. CONCLUSION: Our findings demonstrate that the increase in fatigue which occurs because of excessively elevated brain tryptophan can be further amplified by the use of synthetic KYNA and QUIN.

Sanfilippo B in an elderly female psychiatric patient: a rare but relevant diagnosis in presenile dementia.

OBJECTIVE: Sanfilippo B is a rare autosomal recessive mucopolysaccharidosis (MPS IIIB) caused by a deficiency of N-acetyl-alpha-D-glucosaminidase (NAGLU). METHOD: A mild mentally retarded elderly female patient is described with a slowly progressive dementia who had given birth to a daughter who developed normally. RESULTS: Metabolic screening revealed an enhanced concentration of heparan sulfate in urine. Enzymatic assay demonstrated deficiency of N-acetyl-alpha-D-glucosaminidase. Mutations in the NAGLU gene were found. One mentally retarded and hospitalized elder brother was also found to have MPS IIIB, whereas a second brother, who had died earlier, is suspected to have had the same metabolic disorder. Prior to the development of dementia, both the patient and her brother showed autistic like features, signs of ideomotor apraxia and weakness in verbal comprehension. CONCLUSION: Screening for metabolic disorders, in particular MPSes, should always be considered in patients with a history of mental deficit and dementia or progressive functional decline.

MUCOPOLYSACARIDOSIS TYPE IIIB MISDIAGNOSED AS AN AUTISTIC SPECTRUM DISORDER: A CASE REPORT AND LITERATURE REVIEW.

OBJECTIVE: To report a rare case of mucopolysaccharidosis IIIB in a pediatric patient, with emphasis on the description of the clinical manifestations and the early diagnosis. CASE DESCRIPTION: A 14-year-old male patient, who presented regression of neuropsychomotor development since his three years and six months old, with speech loss and frequent falls, evolving with behavioral changes, with agitation and aggressiveness. Although being diagnosed with autism, there was no response to the established treatment; he was subsequently submitted to metabolic investigation, which lead to the diagnosis of Mucopolysaccharidosis IIIB. COMMENTS: Identifying a metabolic disorder requires connecting multiple signs and symptoms, as well as eliminating other apparent causes. MPS IIIB is a diagnostic challenge, particularly in the early stages and in the absence of a family history of the disease.

Central nervous system pathology in preclinical MPS IIIB dogs reveals progressive changes in clinically relevant brain regions.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

Sanfilippo syndrome: a mini-review.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is an autosomal recessive disorder, caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of the glycosaminoglycan heparan sulfate. Based on the enzyme deficiency, four different subtypes, MPS IIIA, B, C, and D, are recognized. The genes encoding these four enzymes have been characterized and various mutations have been reported. The probable diagnosis of all MPS III subtypes is based on increased concentration of heparan sulfate in the urine. Enzymatic assays in leukocytes and/or fibroblasts confirm the diagnosis and allow for discrimination between the different subtypes of the disease. The clinical course of MPS III can be divided into three phases. In the first phase, which usually starts between 1 and 4 years of age, a developmental delay becomes apparent after an initial normal development during the first 1-2 years of life. The second phase generally starts around 3-4 years and is characterized by severe behavioural problems and progressive mental deterioration ultimately leading to severe dementia. In the third and final stage, behavioural problems slowly disappear, but motor retardation with swallowing difficulties and spasticity emerge. Patients usually die at the end of the second or beginning of the third decade of life, although survival into the fourth decade has been reported. Although currently no effective therapy is yet available for MPS III, several promising developments raise hope that therapeutic interventions, halting the devastating mental and behavioural deterioration, might be feasible in the near future.

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu-/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs. ABBREVIATIONS: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model.

Numerous data support passage of maternal cells into the fetus during pregnancy in both human and animal models. However, functional benefits of maternal microchimerism in utero are unknown. The current study attempted to take advantage of this route for prenatal delivery of alpha-N-acetylglucosaminidase (Naglu) enzyme into the enzyme-deficient mouse model of Sanfilippo syndrome type B (MPS III B). Enzymatically sufficient mononuclear cells from human umbilical cord blood (MNC hUCB) were intravenously administered into heterozygote females modeling MPS III B on the 5th day of pregnancy during blastocyst implantation. The major findings were 1) administered MNC hUCB cells transmigrated and diffused into the embryos (E12.5); 2) some transmigrated cells expressed CD34 and CD117 antigens; 3) transmigrated cells were found in both the maternal and embryonic parts of placentas; 4) transmigrated cells corrected Naglu enzyme activity in all embryos; 5) administered MNC hUCB cells were extensively distributed in the organs and the blood of heterozygote mothers at one week after transplantation. Results indicate that prenatal delivery of Naglu enzyme by MNC hUCB cell administration into mothers of enzyme-deficient embryos is possible and may present a significant opportunity for new biotechnologies to treat many inherited disorders.

The hexosamine signaling pathway: deciphering the "O-GlcNAc code".

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.

Mucopolysacaridosis type iiib misdiagnosed as an autistic spectrum disorder: a case report and literature review / Mucopolissacaridose tipo iii b mal diagnosticada como transtorno de espectro autista: relato de caso e revisão de literatura

ABSTRACT Objective: To report a rare case of mucopolysaccharidosis IIIB in a pediatric patient, with emphasis on the description of the clinical manifestations and the early diagnosis. Case description: A 14-year-old male patient, who presented regression of neuropsychomotor development since his three years and six months old, with speech loss and frequent falls, evolving with behavioral changes, with agitation and aggressiveness. Although being diagnosed with autism, there was no response to the established treatment; he was subsequently submitted to metabolic investigation, which lead to the diagnosis of Mucopolysaccharidosis IIIB. Comments: Identifying a metabolic disorder requires connecting multiple signs and symptoms, as well as eliminating other apparent causes. MPS IIIB is a diagnostic challenge, particularly in the early stages and in the absence of a family history of the disease.

RESUMO Objetivo: Relatar o caso raro de um paciente pediátrico com mucopolissacaridose III B, com ênfase na descrição de manifestações clínicas. Descrição do caso: Paciente masculino de 14 anos que, a partir dos 3 anos e 6 meses de idade, apresentou regressão do desenvolvimento neuropsicomotor, com perda da fala e quedas frequentes, evoluindo com alterações comportamentais, agitação e agressividade. Diagnosticado como autista, não obteve resposta ao tratamento estabelecido, sendo posteriormente submetido à investigação metabólica, que evidenciou o diagnóstico de mucopolissacaridose III B. Comentários: A identificação de um distúrbio metabólico exige conectar vários sinais e sintomas, além de eliminar outras causas aparentes. A mucopolissacaridose III B é um desafio diagnóstico, particularmente nos estágios iniciais e na ausência de história familiar da doença.

Improved behavior and neuropathology in the mouse model of Sanfilippo type IIIB disease after adeno-associated virus-mediated gene transfer in the striatum.

Sanfilippo syndrome is a mucopolysaccharidosis (MPS) caused by a lysosomal enzyme defect interrupting the degradation pathway of heparan sulfates. Affected children develop hyperactivity, aggressiveness, delayed development, and severe neuropathology. We observed relevant behaviors in the mouse model of Sanfilippo syndrome type B (MPSIIIB), in which the gene coding for alpha-N-acetylglucosaminidase (NaGlu) is invalidated. We addressed the feasibility of gene therapy in these animals. Vectors derived from adeno-associated virus serotype 2 (AAV2) or 5 (AAV5) coding for NaGlu were injected at a single site in the putamen of 45 6-week-old MPSIIIB mice. Normal behavior was observed in treated mice. High NaGlu activity, far above physiological levels, was measured in the brain and persisted at 38 weeks of age. NaGlu immunoreactivity was detected in neuron intracellular organelles, including lysosomes. Enzyme activity spread beyond vector diffusion areas. Delivery to the entire brain was reproducibly obtained with both vector types. NaGlu activity was higher and distribution was broader with AAV5-NaGlu than with AAV2-NaGlu vectors. The compensatory increase in the activity of various lysosomal enzymes was improved. The accumulation of gangliosides GM2 and GM3 present before treatment and possibly participating in neuropathology was reversed. Characteristic vacuolations in microglia, perivascular cells, and neurons, which were prominent before the age of treatment, disappeared in areas in which NaGlu was present. However, improvement was only partial in some animals, in contrast to high NaGlu activity. These results indicate that NaGlu delivery from intracerebral sources has the capacity to alleviate most disease manifestations in the MPSIIIB mouse model.

Cell-to-cell contact between normal fibroblasts and lymphoblasts deficient in lysosomal enzymes.

Human lymphoblasts deficient in iduronate sulfatase or in alpha-N-acetylglucosaminidase acquire discrete levels of enzyme activity after co-culture with human normal skin fibroblasts. This occurs by direct cell-to-cell contact and not by uptake of secreted fibroblast enzyme. The process is dependent on time and on the number of fibroblasts used. Electron-microscopic examination of the co-culture of the two cell types reveals extensive region of intimate contact. Fibroblastic projections appear frequently in close apposition with lymphoblast invaginations; a diffuse micropinocytotic activity is evident only in fibroblastic cells.

Mucopolysaccharidosis type IIIB: characterisation and expression of wild-type and mutant recombinant alpha-N-acetylglucosaminidase and relationship with sanfilippo phenotype in an attenuated patient.

Mucopolysaccharidosis type IIIB (MPS-IIB) is a lysosomal storage disorder characterised by the defective degradation of heparan sulfate due to a deficiency of alpha-N-acetylglucosaminidase (NAG). The clinical severity of MPS-IIIB ranges from an attenuated to severely affected Sanfilippo phenotype. This paper describes the expression and characterisation of wild-type recombinant NAG and the molecular characterisation of a previously identified R297X/F48L compound heterozygous MPS-IIIB patient with attenuated Sanfilippo syndrome. We have previously shown R297X to be the most common mutation in a cohort of Dutch and Australian patients, occurring at a frequency of approximately 12.5%. To date F48L has only been described in the proband. To determine the contribution of each mutation to the overall clinical phenotype of the patient, both mutant alleles were engineered into the wild-type NAG cDNA and expressed in Chinese hamster ovary cells. The wild-type NAG and F48L mutant alleles were also retrovirally expressed in MPS-IIIB skin fibroblasts. Residual NAG activity and the stability and maturation of immunoprecipitated NAG were determined for wild-type NAG and mutant NAG. The combined biochemical phenotypes of the two NAG mutant alleles demonstrated a good correspondence with the observed attenuated Sanfilippo phenotype of the patient.

Deficiency of N-acetyl-beta-D-glucosaminidase in neutrophils of cancer patients.

It has been suggested that intracellular enzymatic deficiencies of neutrophils may represent another cancer risk factor. This concept is based on data indicating the cytotoxic effects of neutrophils on tumor cells. In 196 patients with various malignancies, precancerous states and states predisposing to malignancies, N-acetyl-beta-D-glucosaminidase activity has been determined in peripheral blood neutrophils using a semiquantitative cytochemical method. A statistically significant deficiency of this enzyme in neutrophils has been reported in patients with uterine, breast and lung cancer and uterine myoma. A tendency towards lower levels of activity of the enzyme within neutrophils has also been noted in patients with carcinoma of the stomach and precancerous states of the larynx, although without statistical significance; patients with endometriosis showed elevated activity of the enzyme.

Sanfilippo B syndrome (MPS III B): case report with analysis of CSF mucopolysaccharides and conjunctival biopsy.

A case of a child with Sanfilippo B syndrome (MPS III B), born of a consanguineous marriage, is reported. Urinary mucopolysaccharide analysis showed an abnormal excretion mainly of heparan sulphate. N-acetyl-a-glucosaminidase activity was absent in the patient but was present in the heterozygous range in parents and siblings. CSF mucopolysaccharides were also abnormally high. In fibrocytes from conjunctival biopsy and CSF cells numerous vacuoles containing storage material were found. The presence of vacuoles in fibrocytes from conjunctival biopsy and/or in CSF cells can be useful in the diagnosis of many suspected lysosomal storage disorders.

Extraneurologic symptoms as presenting signs of Sanfilippo disease.

Sanfilippo disease, or mucopolysaccharidosis type III, results from the deficiency of lysosomal hydrolases, which impairs heparan sulfate metabolism. Clinically, the disease is characterized by a mild somatic phenotype combined with early severe neurodegenerative illness with prominent behavioral disturbance. We report clinical and molecular findings of a child with Sanfilippo disease type B (alpha-N>-acetylglucosaminidase deficiency) who presented at age 18 months with marked systemic involvement and normal initial psychomotor development. These findings suggest that atypical mucopolysaccharidosis type III patients may present with early somatic changes preceding the onset of overt neurologic symptoms and ensuring an early diagnosis and possible therapeutic intervention.

Mouse model of Sanfilippo syndrome type B: relation of phenotypic features to background strain.

Sanfilippo syndrome type B or mucopolysaccharidosis type III B (MPS IIIB) is a lysosomal storage disorder that is inherited in autosomal recessive manner. It is characterized by systemic heparan sulfate accumulation in lysosomes due to deficiency of the enzyme alpha-N-acetylglucosaminidase (Naglu). Devastating clinical abnormalities with severe central nervous system involvement and somatic disease lead to premature death. A mouse model of Sanfilippo syndrome type B was created by targeted disruption of the gene encoding Naglu, providing a powerful tool for understanding pathogenesis and developing novel therapeutic strategies. However, the JAX GEMM Strain B6.129S6-Naglutm1Efn mouse, although showing biochemical similarities to humans with Sanfilippo syndrome, exhibits aging and behavioral differences. We observed idiosyncrasies, such as skeletal dysmorphism, hydrocephalus, ocular abnormalities, organomegaly, growth retardation, and anomalies of the integument, in our breeding colony of Naglu mutant mice and determined that several of them were at least partially related to the background strain C57BL/6. These background strain abnormalities, therefore, potentially mimic or overlap signs of the induced syndrome in our mice. Our observations may prove useful in studies of Naglu mutant mice. The necessity for distinguishing background anomalies from signs of the modeled disease is apparent.

Diagnosis of the sphingolipidoses with labelled natural substrates.

The genetic heterogeneity of sphingolipidoses is underlined and the desirability of using natural labelled substrates for the diagnoses of each new index case strongly emphasized. Recent studies of our Scandinavian Krabbe families (more than 50) have repeatedly shown that there is no method developed which can be used for the detection of carriers of the mutant gene in leukocytes or lymphocytes. Also described are enzymic studies in two forms of Gaucher disease which further demonstrate the importance of natural substrates for the diagnoses of the disease in leukocytes and cultivated amniotic fluid cells.

Enzymatic deficiencies of the immune system cells in patients with cancer of the larynx and other malignancies.

Activity of N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and acid phosphatase and myeloperoxidase was determined in neutrophils and lymphocytes of patients with cancer of the larynx and precancerous states of the larynx as well as--for comparative reasons--in patients with malignant tumors of female generation organs, breast carcinoma, cancer of the stomach and endometriosis. The main result of investigations performed was in fact that intracellular deficiency of beta-glucuronidase within the neutrophils characterizes patients with cancer and precancerous states of the larynx. Patients with cancer of the larynx show additionally a deficiency of neutrophil myeloperoxidase. Deficiency of N-acetyl-beta-D-glucosaminidase occurs, in contrast, in patients with malignancies of female generation organ. Activity of myeloperoxidase in neutrophils from patients with gastric carcinoma is slightly elevated.