Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4−/−;Hexa−/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa−/− or Neu4−/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating GM2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa−/− mice

Brain pathology in mucopolysaccharidoses (MPS) patients with neurological forms

Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2 ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutral glycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers are similar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice

Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling

The ectodomain of TOLL-like receptors (TLR) is highly glycosylated with several N-linked gylcosylation sites located in the inner concave surface. The precise role of these sugar N-glycans in TLR receptor activation is unknown. Recently, we have shown that Neu1 sialidase and not Neu2, -3 and -4 forms a complex with TLR-2, -3 and -4 receptors on the cell-surfacemembrane of naïve and activated macrophage cells (Glycoconj J DOI 10.1007/ s10719-009-9239-8). Activation of Neu1 is induced by TLR ligands binding to their respective receptors. Here,we show that endotoxin lipopolysaccharide (LPS)-induced MyD88/TLR4 complex formation and subsequent NFκB activation is dependent on the removal ofα-2,3-sialyl residue linked to β-galactoside of TLR4 by theNeu1 activity associated with LPS-stimulated live primary macrophage cells,macrophage and dendritic cell lines but not with primary Neu1-deficient macrophage cells. Exogenous α-2,3 sialyl specific neuraminidase (Streptoccocus pneumoniae) and wild-type T. cruzi trans-sialidase (TS) but not the catalytically inactive mutant TSΔAsp98-Glu mediate TLR4 dimerization to facilitate MyD88/TLR4 complex formation and NFκB activation similar to those responses seen with LPS. These same TLR ligand-induced NFκB responses are not observed in TLR deficient HEK293 cells, but are re-established in HEK293 cells stably transfected with TLR4/MD2, and are significantly inhibited by α-2,3-sialyl specific Maackia amurensis (MAL-2) lectin, α-2,3-sialyl specific galectin-1 and neuraminidase inhibitor Tamiflu but not by α-2,6-sialyl specific Sambucus nigra lectin (SNA). Taken together, the findings suggest that Neu1 desialylation of α-2,3-sialyl residues of TLR receptors enables in removing a steric hinderance to receptor association for TLR activation and cellular signaling

Clinical and genetic spectrum of Sanfilippo type c (MPS IIIC) disease in the Netherlands

Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo C syndrome) is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). We performed a clinical study on 29 Dutch MPS IIIC patients and determined causative mutations in the recently identified HGSNAT gene. Psychomotor development was reported to be normal in all patients during the first year of life. First clinical signs were usually noted between I and 6 years (mean 3.5 years), and consisted of delayed psychomotor development and behavioral problems. Other symptoms included sleeping and hearing problems, recurrent infections, diarrhoea and epilepsy. Two sisters had attenuated disease and did not have symptoms until the third decade. Mean age of death was 34 years (range 25-48). Molecular analysis revealed mutations in both alletes for all patients except one. Altogether 14 different mutations were found: two splice site mutations, one frame shift mutation due to an insertion, three nonsense mutations and eight missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin representing 22.0% and 29.3%, respectively, of the mutant alleles. This study demonstrates that MPS IIIC has a milder course than previously reported and that both severity and clinical course are highly variable even between sibs, complicating prediction of the clinical phenotype for individual patients. A clear phenotype-genotype correlation could not be established, except that the mutations p.G262R and p.S539C were only found in two sisters with late-onset disease and presumably convey a mild phenotype. (C) 2007 Elsevier Inc. All rights reserved

Sanfilippo Syndrome Type C: Mutation Spectrum in the Heparan Sulfate Acetyl-CoA: alpha-Glucosaminide N-Acetyltransferase (HGSNAT) Gene

Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N,acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation. of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, I I insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php? select_db = HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS MC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT. Hum Mutat 30, 918-925, 2009. (C) 2009 Wiley-Liss, Inc

Glucosamine amends CNS pathology in mucopolysaccharidosis IIIC mouse expressing misfolded HGSNAT

The majority of mucopolysaccharidosis IIIC (MPS IIIC) patients have missense variants causing misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), which are potentially treatable with pharmacological chaperones. To test this approach, we generated a novel HgsnatP304L mouse model expressing misfolded HGSNAT Pro304Leu variant. HgsnatP304L mice present deficits in short-term and working/spatial memory 2–4 mo earlier than previously described constitutive knockout Hgsnat-Geo mice. HgsnatP304L mice also show augmented severity of neuroimmune response, synaptic deficits, and neuronal storage of misfolded proteins and gangliosides compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons further reduced levels of synaptic proteins. Memory deficits and majority of brain pathology were rescued in mice receiving HGSNAT chaperone, glucosamine. Our data for the first time demonstrate dominant-negative effects of misfolded HGSNAT Pro304Leu variant and show that they are treatable by oral administration of glucosamine. This suggests that patients affected with mutations preventing normal folding of the enzyme can benefit from chaperone therapy

Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides

Sialylated glycolipids, gangliosides play an essential role in the central nervous system regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in development of severe neurological disorders, gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that two mammalian enzymes, neuraminidases 3 and 4 play important role in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In the neuraminidase 3-4 double knockout mice, GM3 ganglioside is stored in microglia, vascular pericytes and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies and memory loss whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro. Double knockout mice also have reduced levels of GM1 ganglioside and myelin in neuronal axons. Besides, neuraminidase 3 deficiency drastically increased storage of GM2 in the brain tissues of the asymptomatic mouse model of the severe human gangliosidosis, Tay-Sachs disease indicating that this enzyme is responsible for the metabolic bypass of the β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies

Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides

Sialylated glycolipids, gangliosides play an essential role in the central nervous system regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in development of severe neurological disorders, gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that two mammalian enzymes, neuraminidases 3 and 4 play important role in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In the neuraminidase 3-4 double knockout mice, GM3 ganglioside is stored in microglia, vascular pericytes and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies and memory loss whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro. Double knockout mice also have reduced levels of GM1 ganglioside and myelin in neuronal axons. Besides, neuraminidase 3 deficiency drastically increased storage of GM2 in the brain tissues of the asymptomatic mouse model of the severe human gangliosidosis, Tay-Sachs disease indicating that this enzyme is responsible for the metabolic bypass of the β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies