The role of autophagy in the pathogenesis of glycogen storage disease type II (GSDII)

Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid α-glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.     

Towards a molecular therapy for glycogen storage disease type II (Pompe disease)

Glycogen storage disease type II (GSD-II), also known as Pompe disease, is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. Currently, there is no treatment for this fatal disorder. However, several lines of research suggest the possibility of future treatment. Enzyme replacement strategies hold the greatest hope for patients currently affected by GSD-II, but future strategies could include in vivo or ex vivo gene therapy approaches and/or mesenchymal stem cell or bone-marrow transplantation approaches. Each of the approaches might eventually be combined to further improve the overall clinical efficacy of any one treatment regimen. The lessons learned from GSD-II research will also benefit a great number of individuals affected by other genetic disorders.     

A newly identified c.1824_1828dupATACG mutation in exon 13 of the GAA gene in infantile-onset glycogen storage disease type II (Pompe disease)

Pompe disease or glycogen storage disease type II is a glycogen storage disorder associated with malfunction of the acid α-glucosidase enzyme (GAA; EC.3.2.1.3) leading to intracellular aggregations of glycogenin muscles. The infantile-onset type is the most life-threatening form of this disease, in which most of patients suffer from cardiomyopathy and hypotonia in early infancy. In this study, a typical case of Pompe disease was reported in an Iranian patient using molecular analysis of the GAA gene. Our results revealed a new c.1824_1828dupATACG mutation in exon 13 of the GAA gene. In conclusion, with the finding of this novel mutation, the genotypic spectrum of Iranian patients with Pompe disease has been extended, facilitating the definition of disease-related mutations.     

The polymorphic locus for glycogen storage disease VI (liver glycogen phosphorylase) maps to chromosome 14.

Human liver glycogen phosphorylase deficiency, also known as glycogen storage disease type VI (GSD VI) or Hers disease, is characterized by hepatomegaly and reduced or absent glycogenolytic response to the injection of glucagon. The recently isolated cDNA encoding the liver isozyme of glycogen phosphorylase was used to map the gene and identify restriction-fragment polymorphisms in normal Caucasians as a prerequisite for detecting linked GSD VI abnormalities. Results of restriction-enzyme analysis using a downstream fragment of the liver glycogen phosphorylase cDNA indicated the existence of a single gene copy per haploid genome. Hybridization of this downstream liver phosphorylase probe to dual laser-excited, sorted human chromosomes localized the gene to human chromosome 14. When the downstream probe was tested on genomic DNA cut with seven different restriction enzymes, a single MspI restriction-fragment-length polymorphism (RFLP) was observed in a single individual. In contrast, similar Southern blots performed with an upstream portion of the cDNA encoding liver phosphorylase revealed common RFLPs for four of eight enzymes tested, with minor polymorphic allele frequencies ranging from 33% to 44%. One of the four enzymes (TaqI) revealed two independent polymorphisms. If random distribution of these haplotypes among normal and disease loci, is assumed, approximately 92% of fetuses at risk for Hers disease will be informative when tested with the upstream liver phosphorylase probe.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Gaucher disease (Norrbottnian type III): probable founders identified by genealogical and molecular studies

Gaucher disease type III (GD) is found at a high frequency in northern Sweden. The contemporary Swedish index families are found in two geographically distinct clusters with the highest world-wide frequency of type III GD. A single T-to-C transition in exon 10 has previously been identified in patients from one of the two isolates and we report there the same mutation in the second isolate. Mutational analysis was combined with a genealogical reconstruction of 19 contemporary index families. Both clusters were traced back to two corresponding pairs of ancestors over a 9–13 generation span. Molecular studies show that the two clusters are compatible with a single founder who arrived in northern Sweden in or before the 16th century.     

Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.     

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

On the molecular basis linking Nerve Growth Factor (NGF) to Alzheimer's disease

1. Alzheimer's disease (AD) is pathologically defined by the deposition of amyloid peptide and neurofibrillary tangles and is characterized by a progressive loss of cognition and memory function, due to marked cortical cholinergic depletion. 2. Cholinergic cortical innervation is provided by basal forebrain cholinergic neurons. The neurotrophin Nerve Growth Factor (NGF) promotes survival and differentiation of basal forebrain cholinergic neurons. 3. This assertion has been at the basis of the hypothesis developed in the last 20 years, whereby NGF deprivation would be one of the factor involved in the etiology of sporadic forms of AD. 4. In this review, we shall summarize data that lead to the production and characterization of a mouse model for AD (AD11 anti-NGF mice), based on the expression of transgenic antibodies neutralizing NGF. The AD-like phenotype of AD11 mice will be discussed on the basis of recent studies that have posed NGF and its precursor pro-NGF back to the stage of AD-like neurodegeneration, showing the involvement of the precursor pro-NGF in one of the cascades leading to AD neurodegeneration.     

Palladium(II) and platinum(II) monoselenocarboxylates: Synthesis, spectroscopy, structure and their use as molecular precursors for metal selenides

Treatment of MCl₂(P^∩P) or MCl₂(PⁿPr₃)₂ with two equivalents of ArCOSeK readily yields cis-[M(SeCOAr)₂(P^∩P)] and trans-[M(SeCOAr)₂(PⁿPr₃)₂], respectively (M = Pd or Pt; Ar = Ph or 4-MeC₆H₄; P^∩P = dppm, dppe, dppp). The reaction of Pd(SeCOAr)₂(dppe) with PdCl₂(dppe) in the presence of NaBPh₄ in methanol gave a tri-nuclear ionic complex, [Pd₃(μ₃-Se)₂(dppe)₃][BPh₄]₂. These complexes were characterized by UV-Vis, IR and NMR spectroscopy. The complex [Pt(SeCOPh)₂(dppp)] has been structurally characterized by X-ray crystallography. The coordination environment around square planar platinum atom is defined by chelating dppp ligand and two unidentate selenocarboxylates bonded through selenium atoms. Pyrolysis of [Pd(SeCOAr)₂(PⁿPr₃)₂] either in tri-n-butylphosphate (TBP) (at 200 °C) or in the solid state (furnace heating at 350 °C) gave Pd₁₇Se₁₅.     

A multiplex PCR assay for molecular sexing of the naked mole-rat (Heterocephalus glaber)

For molecular sexing of the naked mole-rat (Heterocephalus glaber), we designed a PCR primer set to amplify part of the Y-linked DBY gene. When this primer set was applied to the samples of known sex with the 16S rRNA gene (16S rDNA) primers as control, PCR products were successfully obtained as two DNA bands in males, a male-specific 163 bp DBY band and a 446 bp band of 16S rDNA shared with females, whereas females showed only the common band. This result shows that this multiplex PCR assay is useful for sex identification of H. glaber.     

A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) is a purine salvage enzyme that catalyses the conversion of hypoxanthine and guanine to their respective mononucleotides. Partial deficiency of this enzyme can result in the overproduction of uric acid leading to a severe form of gout, whilst a virtual absence of HPRT activity causes the Lesch-Nyhan syndrome which is characterised by hyperuricaemia, mental retardation, choreoathetosis and compulsive self-mutilation. The HPRT-encoding gene is located on the X chromosome in the region q₂₆–q₂₇ and consists of nine exons and eight introns totalling 57 kb. This gene is transcribed to produce an mRNA of 1.6 kb, which contains a protein encoding region of 654 nucleotides. With the advent of increasingly refined techniques of molecular biology, it has been possible to study the HPRT gene of individuals with a deficiency in HPRT activity to determine the genetic basis of the enzyme deficiency. Many different mutations throughout the coding region have been described, but in the absence of precise information on the three-dimensional structure of the HPRT protein, it remains difficult to determine any consistent correlation between the structure and function of the enzyme.     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

A new peptidic vector for molecular imaging of apoptosis, identified by phage display technology

Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app) for PS ranging from 6.08x10(10) M to 1.62x10(11)M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app=(1.6+/-0.2)x10(11)M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction.     

Partial deletion of the X chromosome in gonadal dysgenesis 46,X,del(X)(p22) identified by BUdR treatment

Summary In this report we describe a deletion of the short arm of the X chromosome in a 16-year-old female with gonadal dysgenesis.The breakpoint was localized by BUdR treatment and acridine orange staining in region 2, band 2.Of the examined cells, 3% showed an early replication of the deleted X chromosome.