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Journal of Neurochemistry Jun 2010Sandhoff disease is an autosomal recessive, neurodegenerative disease involving the storage of brain ganglioside GM2 and asialo-GM2. Previous studies showed that caloric...
Sandhoff disease is an autosomal recessive, neurodegenerative disease involving the storage of brain ganglioside GM2 and asialo-GM2. Previous studies showed that caloric restriction, which augments longevity, and N-butyldeoxynojirimycin (NB-DNJ, Miglustat), an imino sugar that hinders the glucosyltransferase catalyzing the first step in glycosphingolipid biosynthesis, both increase longevity and improve motor behavior in the beta-hexosaminidase (Hexb) knockout (-/-) murine model of Sandhoff disease. In this study, we used a restricted ketogenic diet (KD-R) and NB-DNJ to combat ganglioside accumulation. Adult Hexb-/- mice were placed into one of the following groups: (i) a standard diet (SD), (ii) a SD with NB-DNJ (SD + NB-DNJ), (iii) a KD-R, and (iv) a KD-R with NB-DNJ (KD-R + NB-DNJ). Forebrain GM2 content (mug sialic acid/100 mg dry wt) in the four groups was 375 +/- 15, 312 +/- 8, 340 +/- 28, and 279 +/- 26, respectively, indicating an additive interaction between NB-DNJ and the KD-R. Most interestingly, brain NB-DNJ content was 3.5-fold greater in the KD-R + NB-DNJ mice than in the SD + NB-DNJ mice. These data suggest that the KD-R and NB-DNJ may be a potential combinatorial therapy for Sandhoff disease by enhancing NB-DNJ delivery to the brain and may allow lower dosing to achieve the same degree of efficacy as high dose monotherapy.
Topics: 1-Deoxynojirimycin; 3-Hydroxybutyric Acid; Analysis of Variance; Animals; Blood Glucose; Body Weight; Brain; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diet, Ketogenic; Eating; G(M2) Ganglioside; Lipid Metabolism; Mice; Mice, Knockout; Myelin Sheath; Purkinje Cells; Sandhoff Disease; beta-N-Acetylhexosaminidases
PubMed: 20374428
DOI: 10.1111/j.1471-4159.2010.06733.x -
Neurology Jan 2010To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS).
OBJECTIVE
To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS).
METHODS
We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples.
RESULTS
We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L).
CONCLUSION
In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.
Topics: Child; Child, Preschool; Cross-Sectional Studies; Demyelinating Diseases; Dipeptides; Female; Genetic Testing; Genotype; Humans; Infant; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Male; Mutation; N-Acetylneuraminic Acid; Organic Anion Transporters; Sialic Acid Storage Disease; Symporters; Young Adult
PubMed: 20101035
DOI: 10.1212/WNL.0b013e3181cbcdc4 -
The Journal of Neuroscience : the... Dec 2009Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane...
Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane protein that transports free sialic acid out of the lysosome after it is cleaved from sialoglycoconjugates undergoing degradation. Accumulation of sialic acid in lysosomes defines these disorders, and the clinical phenotype is characterized by neurodevelopmental defects, including severe CNS hypomyelination. In this study, we used a sialin-deficient mouse to address how loss of sialin leads to the defect in myelination. Behavioral analysis of the sialin(-/-) mouse demonstrates poor coordination, seizures, and premature death. Analysis by histology, electron microscopy, and Western blotting reveals a decrease in myelination of the CNS but normal neuronal cytoarchitecture and normal myelination of the PNS. To investigate potential mechanisms underlying CNS hypomyelination, we studied myelination and oligodendrocyte development in optic nerves. We found reduced numbers of myelinated axons in optic nerves from sialin(-/-) mice, but the myelin that was present appeared grossly normal. Migration and density of oligodendrocyte precursor cells were normal; however, a marked decrease in the number of postmitotic oligodendrocytes and an associated increase in the number of apoptotic cells during the later stages of myelinogenesis were observed. These findings suggest that a defect in maturation of cells in the oligodendrocyte lineage leads to increased apoptosis and underlies the myelination defect associated with sialin loss.
Topics: Animals; Apoptosis; Axons; Brain; Cell Count; Cell Movement; Longevity; Mice; Mice, Knockout; Motor Activity; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Neurons; Oligodendroglia; Optic Nerve; Organic Anion Transporters; Peripheral Nervous System; Seizures; Spinal Cord; Stem Cells; Symporters; Transcription Factors
PubMed: 20007460
DOI: 10.1523/JNEUROSCI.3005-09.2009 -
Glycobiology Mar 2010The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the...
The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. So far, no active site geometry predictions or conformational transitions involved with function are available for mammalian GNE/MNK. The N-terminal GNE domain is homologous to various prokaryotic 2-epimerases, some of which have solved crystallographic structures. The C-terminal MNK domain belongs to the sugar kinases superfamily; its crystallographic structure is solved at 2.84 A and three-dimensional structures have also been reported for several other kinases. In this work, we employed available structural data of GNE/MNK homologs to model the active sites of human GNE/MNK and identify critical amino acid residues responsible for interactions with substrates. In addition, we modeled effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action. We found that all reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK. The Persian-Jewish HIBM founder mutation p.M712T is located at the interface alpha4alpha10 and likely affects GlcNAc, Mg2+, and ATP binding. This work contributes to further understanding of GNE/MNK function and ligand binding, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.
Topics: Amino Acid Sequence; Humans; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Muscular Diseases; Mutation; N-Acetylneuraminic Acid; Protein Conformation; Sialic Acid Storage Disease
PubMed: 19917666
DOI: 10.1093/glycob/cwp176 -
Annals of Neurology Jun 2009We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of... (Comparative Study)
Comparative Study
We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.
Topics: Adolescent; Child; Diagnosis, Differential; Hereditary Central Nervous System Demyelinating Diseases; Humans; N-Acetylneuraminic Acid; Nuclear Magnetic Resonance, Biomolecular; Organic Anion Transporters; Sialic Acid Storage Disease; Symporters; Young Adult
PubMed: 19557856
DOI: 10.1002/ana.21624 -
Brain : a Journal of Neurology Mar 2009In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton...
In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Atrophy; Cells, Cultured; Cerebellar Ataxia; Cerebellum; Child; Child, Preschool; Cohort Studies; Female; Humans; Infant; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Male; Middle Aged; N-Acetylneuraminic Acid; Transferrin
PubMed: 19153153
DOI: 10.1093/brain/awn355 -
Biochimica Et Biophysica Acta Apr 2009Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and... (Review)
Review
Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and fusion with other organelles. This review summarizes the molecular mechanisms of lysosomal storage diseases caused by defective transport of small molecules or ions across the lysosomal membrane, as well as Danon disease. In cystinosis and free sialic acid storage diseases, transporters for cystine and acidic monosaccharides, respectively, are blocked or retarded. A putative cobalamin transporter and a hybrid transporter/transferase of acetyl groups are defective in cobalamin F type disease and mucopolysaccharidosis type IIIC, respectively. In neurodegenerative forms of osteopetrosis, mutations of a proton/chloride exchanger impair the charge balance required for sustained proton pumping by the V-type ATPase, thus resulting in bone-resorption lacuna neutralization. However, the mechanism leading to lysosomal storage and neurodegeneration remains unclear. Mucolipidosis type IV is caused by mutations of a lysosomal cation channel named TRPML1; its gating properties are still poorly understood and the ion species linking this channel to lipid storage and membrane traffic defects is debated. Finally, the autophagy defect of Danon disease apparently arises from a role of LAMP2 in lysosome/autophagosome fusion, possibly secondary to a role in dynein-based centripetal motility.
Topics: Animals; Autophagy; Biological Transport; Humans; Lysosomal Storage Diseases; Lysosomes; Membrane Proteins; Proteins
PubMed: 19146888
DOI: 10.1016/j.bbamcr.2008.12.008 -
Proceedings of the National Academy of... Aug 2008Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes...
Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H(+)/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H(+)/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.
Topics: Adenosine Triphosphate; Amino Acid Transport Systems, Acidic; Animals; Aspartic Acid; Biological Transport; Cells, Cultured; Exocytosis; Glutamic Acid; Hippocampus; Humans; Mice; N-Acetylneuraminic Acid; Neurons; Organic Anion Transporters; Phylogeny; Rats; Rats, Wistar; Symporters
PubMed: 18695252
DOI: 10.1073/pnas.0804015105 -
FASEB Journal : Official Publication of... Nov 2008Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused...
Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.
Topics: Alleles; Amino Acid Substitution; Cells, Cultured; Cytidine Monophosphate N-Acetylneuraminic Acid; Fibroblasts; Genes, Dominant; Humans; Multienzyme Complexes; Mutation, Missense; RNA Interference; RNA, Small Interfering; Sialic Acid Storage Disease
PubMed: 18653764
DOI: 10.1096/fj.08-110890 -
Gene Regulation and Systems Biology Jun 2008Hereditary Inclusion Body Myopathy (HIBM2) is a chronic progressive skeletal muscle wasting disorder which generally leads to complete disability before the age of 50...
Hereditary Inclusion Body Myopathy (HIBM2) is a chronic progressive skeletal muscle wasting disorder which generally leads to complete disability before the age of 50 years. There is currently no effective therapeutic treatment for HIBM2. Development of this disease is related to expression in family members of an autosomal recessive mutation of the GNE gene, which encodes the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). This is the rate limiting bifunctional enzyme that catalyzes the first 2 steps of sialic acid biosynthesis. Decreased sialic acid production, consequently leads to decreased sialyation of a variety of glycoproteins including the critical muscle protein alpha-dystroglycan (alpha-DG). This in turn severely cripples muscle function and leads to the onset of the syndrome. We hypothesize that replacing the mutated GNE gene with the wildtype gene may restore functional capacity of GNE/MNK and therefore production of sialic acid, allowing for improvement in muscle function and/or delay in rate of muscle deterioration. We have constructed three GNE gene/CMV promoter plasmids (encoding the wildtype, HIBM2, and Sialuria forms of GNE) and demonstrated enhanced GNE gene activity following delivery to GNE-deficient CHO-Lec3 cells. GNE/MNK enzyme function was significantly increased and subsequent induction of sialic acid production was demonstrated after transfection into Lec3 cells with the wild type or R266Q mutant GNE vector. These data form the foundation for future preclinical and clinical studies for GNE gene transfer to treat HIBM2 patients.
PubMed: 19787087
DOI: 10.4137/grsb.s728