Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n ‫؍‬ 100) and 1997 (n ‫؍‬ 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp713Val in one, Thr823Ile in six, and Ala1183Thr in one) or in gyrB (six mutations, amino acid changes Asp4263Asn in five and Arg4473Leu in one). These changes are similar to those already described in other species except for Asp713Val, which is novel, and Ala1183Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.Vancomycin and metronidazole are effective antibiotics for the treatment of Clostridium difficile-associated diarrhea (19,24). However, at least 15% of patients relapse after discontinuation of treatment for poorly understood reasons (23, 24). Other treatments may be more effective in preventing relapses.The in vitro activities against C. difficile of older fluoroquinolones like ciprofloxacin and ofloxacin are poor, but those of some new compounds like moxifloxacin are markedly increased (1,3,17,25). Thus, these drugs could become therapeutic alternatives for the treatment of C. difficile-associated diarrhea. Decreased susceptibility to fluoroquinolones in C. difficile has already been described (2, 3, 13, 26), but the data reported are controversial and the genetic mechanisms involved are poorly understood.In the present work, 198 French clinical strains of C. difficile were screened for decreased susceptibility to fluoroquinolones, and the genetic basis of this phenomenon was investigated. MATERIALS AND METHODSBacterial strains. Reference strain ATCC 9689 and 198 nonrepetitive clinical isolates obtained in 1991 (n ϭ 100) and 1997 (n ϭ 98) from 36 French hospitals (7) were studied. Serotyping of the strains had been performed in a previous study (7) by the method of Delmée et al. (12). Serogroups A, C, D, F, G, H, and K accounted for 9, 22, 8, 1, 4, 19, and 14% of the strains, respectively, and 23% of the strains were untypeable. Strains were cultured in...

Tordo et al. present a novel AAV gene therapy vector, AAV-TT, which exceeds the current benchmark neurotropic serotypes AAV9 and AAVrh10 and enables unprecedented correction of a lysosomal transmembrane enzyme deficiency. AAV-TT based gene therapies may thus be suitable for the treatment of human neurological diseases characterised by global neuropathology.

Neuraminidases (sialidases) catalyze the removal of sialic acid residues from sialylated glycoconjugates. We now report that mammalian neuraminidase 1 (Neu1), in addition to its catabolic function in lysosomes, is transported to the cell surface where it is involved in the regulation of insulin signaling. Insulin binding to its receptor rapidly induces interaction of the receptor with Neu1, which hydrolyzes sialic acid residues in the glycan chains of the receptor and, consequently, induces its activation. Cells from sialidosis patients with a genetic deficiency of Neu1 show impairment of insulin-induced phosphorylation of downstream protein kinase AKT, and treatment of these cells with purified Neu1 restores signaling. Genetically modified mice with ∼10% of the normal Neu1 activity exposed to a high-fat diet develop hyperglycemia and insulin resistance twice as fast as their wild-type counterparts. Together, these studies identify Neu1 as a novel component of the signaling pathways of energy metabolism and glucose uptake.

Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis. Several clinically isolated C. difficile strains are resistant to antibiotics other than metronidazole and vancomycin. Recently, bacteriocins of lactic acid bacteria have been proposed as an alternative or complementary treatment. The aim of this study was to investigate the inhibitory effect of nisin, a bacteriocin produced by several strains of Lactococcus lactis, against clinical isolates of C. difficile. Nisin Z obtained from culture of L. lactis subsp. lactis biovar. diacetylactis was tested along with commercial nisin A. The effect of nisin A on C. difficile spores was also examined. Nisin A and Z both inhibited the growth of all C. difficile isolates, and MICs were estimated at 6.2 mg ml 21 for nisin Z and 0.8 mg ml 21 for nisin A. In addition, C. difficile spores were also susceptible to nisin A (25.6 mg ml 21 ), which reduced spore viability by 40-50 %. These results suggested that nisin and hence nisin-producing Lactococcus strains could be used to treat C. difficile-associated diarrhoea. INTRODUCTIONClostridium difficile, a Gram-positive anaerobic sporeforming bacterium, is an emerging pathogen capable of causing severe gastrointestinal illness in individuals undergoing antibiotic therapy (Kelly & LaMont, 2008;Rupnik et al., 2009;Stanley et al., 2013). Infection with C. difficile may produce a wide spectrum of outcomes that range from asymptomatic colonization to acute diarrhoea and pseudomembranous colitis, which can result in colonic perforation and death if untreated (Kelly & LaMont, 2008;Rupnik et al., 2009;Stanley et al., 2013). Whilst the vegetative form of C. difficile is responsible for producing the toxins that cause illness, the spore is the principal transmitted form (Sorg & Sonenshein, 2008). Many antibiotics have been implicated in C. difficile-associated diarrhoea, including clindamycin, ampicillin and amoxicillin, as well as the cephalosporins and fluoroquinolones (Wiström et al., 2001;Stevens et al., 2011;Slimings & Riley, 2014) Current treatment for C. difficile-associated diarrhoea is limited mainly to administration of the antibiotics metronidazole or vancomycin (Surawicz et al., 2013). Furthermore, treatment failure and recurrence of infection have also been reported in 2-38 and 8-50 % of cases, respectively (Aslam et al., 2005). Based on its in vitro activity, its efficacy by either the oral or the intravenous route and its low cost, metronidazole was commonly used for treatment of mild C. difficile infection. However, some studies reported the emergence of C. difficile isolates with a reduced susceptibility to metronidazole (Baines et al., 2008; Peláez et al., 2008). Vancomycin is considered a second-line therapy, as prolonged treatment is associated with higher risk of selecting vancomycin-resistant Enterococcus in addition to its high cost.It is clear that the health sector would benefit from efficacious alternative...

BackgroundMutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.MethodsIn this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.ConclusionsWe have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0180-y) contains supplementary material, which is available to authorized users.

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.

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan‐α‐glucosaminide N‐acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease‐causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice‐site mutation c.494−1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372−2G>A, and c.234+1G>A present in cis with c.564‐98T>C and c.710C>A rare single‐nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease‐causing variants, and reveal the evolutionary history of MPSIIIC.