Guglielmo R. D. Villani

Cytokines, neurotrophins, and oxidative stress in brain disease from mucopolysaccharidosis IIIB.

Mucopolysaccharidosis IIIB (MPS IIIB; Sanfilippo syndrome type B) is characterized by profound neurological deterioration. Because a murine model of MPS IIIB disease is available, we focused on analysis of gene expression in the brain and cerebellum of 7‐month‐old MPS IIIB mice by pathway‐specific filter microarrays designed to probe apoptotic‐related, neurotrophic signalling molecules and inflammatory cytokines and receptors. Moreover, we extended the analysis with real‐time PCR performed at 1, 3, 7 months after birth. Bdnf was down‐regulated in the brain but up‐regulated in the cerebellum at 7 months of age, both at RNA and at protein levels. Cbln1 presented a threefold increase in the oldest brains while remaining unaltered in the cerebellum. Ccl3, Casp11, gp91phox, p67phox, and p47phox showed an increased expression in both brain and cerebellum at each examined time point. Ccl3, in particular, exhibited in both organs and at all times tested approximately a tenfold increase in its expression. Insofar as p47phox, p67phox, and gp91phox are all components of the phagocyte NADPH oxidase, our results suggest the possible involvement of the reactive oxygen species in the genesis of neurodegeneration in MPS IIIB disease.

Treatment of the mouse model of mucopolysaccharidosis type IIIB with lentiviral-NAGLU vector.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (alpha-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8-10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.

Identification of molecular defects in Italian Sanfilippo A patients including 13 novel mutations

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by the deficiency of the enzyme heparin sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. Patients develop central nervous system degeneration resulting in progressive dementia, developmental delay, hyperactivity, and aggressive behaviour; subjects may present a wide spectrum of clinical severity. Here, we report the results from molecular analysis of 24 Italian MPS IIIA patients diagnosed over the last 15 years in our laboratory. Altogether, we were able to characterize 38 out of the 48 (79%) pathogenic alleles. We identified 16 molecular defects, 13 novel. The majority of alterations were missense mutations: on exon two (Y40N; A44T; S66W; R74C), on exon four (G122R; P128L; L146P; R150Q), on exon five (D179N; R182C), on exon six (P227R) and on exon eight (E369K; R377C). Single base pair deletions: on exon two (A52nt‐1) and on exon eight (T360nt‐1) and one base pair insertion on exon eight (V361nt + 1) were also identified. Restriction enzyme or ARMS analyses were used to confirm each alteration. S66W represents the most common alteration in our patients population accounting for 33% of the total alleles. Interestingly, all six patients from Sardinia present this mutation, and five of them are homozygous for this change, suggesting that these subjects may have been derived from a common founder. Hum Mutat 11:313–320, 1998.

Mucopolysaccharidosis IIIB: oxidative damage and cytotoxic cell involvement in the neuronal pathogenesis.

Sanfilippo B syndrome (Mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease due to mutations in the gene encoding alpha-N-acetylglucosaminidase and is characterized by a severe neurological disorder. Although several studies have been reported for the murine model of the disease, the molecular basis and the sequence of events leading to neurodegeneration remain to be clarified. We previously suggested the possible involvement of the reactive oxygen species in the disease pathogenesis. In the present paper we extended the analysis of oxidative stress by evaluating the production of superoxide ions throughout the CNS and by evaluating the effect of the stress on the cellular macromolecules. These approaches applied to one-month-old, three-month-old and six-month-old mice revealed that oxidative stress is present in the affected cerebrum and cerebellum tissues from one month from birth, and that it results primarily in protein oxidation, both in the cerebrum and cerebellum, with lipid peroxidation, and especially DNA oxidation, appearing milder and restricted essentially to the cerebellum. We also identified additional genes possibly associated with the neuropathology of MPS IIIB disease. Real time RT-PCR analysis revealed an altered expression of the Sod1, Ret, Bmp4, Tgfb, Gzmb and Prf1 genes. Since Gzmb and Prf1 are proteins secreted by NK/cytotoxic T-cells, these data suggest the involvement of cytotoxic cells in the neuronal pathogenesis. Extending our previous study, findings reported in the present paper show that oxidative stress and all the analyzed stress-related pathological changes occur very early in the disease course, most likely before one month of age.

Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

Mutations among Italian mucopolysaccharidosis type I patients

A group of 27 Italian patients was screened for α-L-iduronidase mucopolysaccharidosis type I mutations. Mutations were found in 18 patients, with 28 alleles identified. The two most common mutations in northern Europeans (W402X and Q70X) accounted for 11% and 13% of the alleles, respectively. The R89Q mutation, uncommon in Europeans, was found only in one patient, accounting for 1 of 54 alleles (1.9%). The other mutations, P533R, A327P and G51D, accounted for 11%, 5.6% and 9.3% of the total alleles, respectively. Interestingly, the high frequency of the P533R mutation seems to be confined to Sicily and is higher than the 3% reported in a British/Australian study.

Intracranial gene delivery of LV-NAGLU vector corrects neuropathology in murine MPS IIIB†‡

Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha‐N‐acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral‐NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain‐derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation‐ and oxidative stress‐related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response‐to‐therapy biomarker for MPS IIIB.

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Molecular defects in the α-N-acetylglucosaminidase gene in Italian Sanfilippo type B patients

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G→A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G→A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.

Effect of avarol and avarone on in vitro-induced microsomal lipid peroxidation

Lipid peroxidation was employed as an experimental model to study the antioxidant properties of avarol, a sesquiterpenoid hydroquinone and of its quinone, avarone. In the NADPH- or ascorbate-linked lipid peroxidation, avarol and avarone were shown to be more effective as inhibitors than in the t-BuOOH-dependent peroxidative process. However, in all three systems employed avarol was a more powerful inhibitor than avarone. The chemical structure of avarol, having an easily donatable hydrogen atom and its kinetics of inhibition suggested that the hydroquinone acted mainly as a radical scavenger. Conversely avarone appeared to interfere mainly with the initiation phase of lipid peroxidation. However, avarol and the semiquinone intermediate may contribute to the inhibitory action of the quinone. In fact avarone reduction to avarol has been shown to occur in the presence of reducing agents such as ascorbate or Fe(II) and to be catalyzed by NADPH-supplemented microsomes.