Daniela Zizioli

Overexpression of cytosolic sialidase Neu2 induces myoblast differentiation in C2C12 cells.

Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady‐state levels and in the cytosolic sialidase enzymatic activity. To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin‐positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was on the contrary observed in parental and mock‐transfected cells under the same experimental conditions. The results indicate that Neu2 upregulation per se is sufficient to trigger myoblast differentiation in C2C12 cells.

Biosafe inertization of municipal solid waste incinerator residues by COSMOS technology

Municipal solid waste incinerator (MSWI) residues can generate negative environmental impacts when improperly handled. The COlloidal Silica Medium to Obtain Safe inert (COSMOS) technology represents a new method to stabilize MSWI residues and to produce inert safe material. Here we report the results about aquatic biotoxicity of lixiviated MSWI fly ash and the corresponding inertized COSMOS material using a zebrafish (Danio rerio) embryo toxicity test. Quantitative assessment of waste biotoxicity included evaluation of mortality rate and of different morphological and teratogenous endpoints in zebrafish embryos exposed to tested materials from 3 to 72h post-fertilization. The results demonstrate that lixiviated MSWI fly ash exerts a dose-dependent lethal effect paralleled by dramatic morphological/teratogenous alterations and apoptotic events in the whole embryo body. Similar effects were observed following MSWI fly ash stabilization in classical concrete matrices, demonstrating that the obtained materials are not biologically safe. On the contrary, no significant mortality and developmental defects were observed in zebrafish embryos exposed to COSMOS inert solution. Our results provide the first experimental in vivo evidence that, in contrast with concrete stabilization procedure, COSMOS technology provides a biologically safe inert.

Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development.

Down-regulation of coasy, the gene associated with NBIA-VI, reduces Bmp signaling, perturbs dorso-ventral patterning and alters neuronal development in zebrafish

Mutations in Pantothenate kinase 2 and Coenzyme A (CoA) synthase (COASY), genes involved in CoA biosynthesis, are associated with rare neurodegenerative disorders with brain iron accumulation. We showed that zebrafish pank2 gene plays an essential role in brain and vasculature development. Now we extended our study to coasy. The gene has high level of sequence identity with the human ortholog and is ubiquitously expressed from the earliest stages of development. The abrogation of its expression led to strong reduction of CoA content, high lethality and a phenotype resembling to that of dorsalized mutants. Lower doses of morpholino resulted in a milder phenotype, with evident perturbation in neurogenesis and formation of vascular arborization; the dorso-ventral patterning was severely affected, the expression of bone morphogenetic protein (Bmp) receptors and activity were decreased, while cell death increased. These features specifically correlated with the block in CoA biosynthesis and were rescued by the addition of CoA to fish water and the overexpression of the human wild-type, but not mutant gene. These results confirm the absolute requirement for adequate levels of CoA for proper neural and vascular development in zebrafish and point to the Bmp pathway as a possible molecular connection underlining the observed phenotype.

Analysis of three μ1‐AP1 subunits during zebrafish development

Background: The family of AP‐1 complexes mediates protein sorting in the late secretory pathway and it is essential for the development of mammals. The ubiquitously expressed AP‐1A complex consists of four adaptins γ1, β1, μ1A, and σ1A. AP‐1A mediates protein transport between the trans‐Golgi network and early endosomes. The polarized epithelia AP‐1B complex contains the μ1B‐adaptin. AP‐1B mediates specific transport of proteins from basolateral recycling endosomes to the basolateral plasma membrane of polarized epithelial cells. Results: Analysis of the zebrafish genome revealed the existence of three μ1‐adaptin genes, encoding μ1A, μ1B, and the novel isoform μ1C, which is not found in mammals. μ1C shows 80% sequence identity with μ1A and μ1B. The μ1C expression pattern largely overlaps with that of μ1A, while μ1B is expressed in epithelial cells. By knocking‐down the synthesis of μ1A, μ1B and μ1C with antisense morpholino techniques we demonstrate that each of these μ1 adaptins is essential for zebrafish development, with μ1A and μ1C being involved in central nervous system development and μ1B in kidney, gut and liver formation. Conclusions: Zebrafish is unique in expressing three AP‐1 complexes: AP‐1A, AP‐1B, and AP‐1C. Our results demonstrate that they are not redundant and that each of them has specific functions, which cannot be fulfilled by one of the other isoforms. Each of the μ1 adaptins appears to mediate specific molecular mechanisms essential for early developmental processes, which depends on specific intracellular vesicular protein sorting pathways. Developmental Dynamics 243:299–314, 2014.

Characterization and expression analysis of mcoln1.1 and mcoln1.2, the putative zebrafish co-orthologs of the gene responsible for human mucolipidosis type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene coding for mucolipin-1 (TRPML1). TRPML1 belongs to a transient receptor potential channels (TRP) subfamily, which in mammals includes two other members: mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). Bioinformatic analysis of the Danio rerio (zebrafish) genome and trascriptome revealed the presence of five different genes related to human mucolipins: mcoln1.1, mcoln1.2, mcoln2, mcoln3.1 and mcoln3.2. We focused our efforts on the characterization of the two putative zebrafish MCOLN1 co-orthologs. Transient-expression experiments in human HeLa cells demonstrated that fish Mcoln1.1 and Mcoln1.2, similarly to TRPML1, localize to late endosomal/lysosomal compartments. Real-Time PCR (RT-PCR) experiments showed that both genes are maternally expressed and transcribed at different levels during embryogenesis. RT-PCR analysis in different zebrafish tissues displayed ubiquitary expression for mcoln1.1 and a more tissue-specific pattern for mcoln1.2. Spatial and temporal expression studies using whole-mount in situ hybridization confirmed that both genes are maternally expressed and ubiquitously transcribed during gastrulation and early somitogenesis. Notably, in the next developmental stages they are more expressed in neural regions and in retina layers, tissues affected in MLIV. Interestingly, mcoln1.1 is detected, from 10 somite-stage until to 36 hpf, in the yolk syncytial layer (YSL) and in the intermediate cell mass (ICM), the earliest site of hematopoiesis. Overall, the redundancy of mucolipins together with their expression profile support the biological relevance of this class of proteins in zebrafish. The data herein presented indicate that Danio rerio could be a suitable vertebrate model for the study of some aspects of MLIV pathogenesis.

Cytosolic sialidase from pig brain: a ‘protein complex’ containing catalytic and protective units☆

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.

Molecular cloning and knockdown of galactocerebrosidase in zebrafish: New insights into the pathogenesis of Krabbe's disease

The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbes disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.

slc7a6os Gene Plays a Critical Role in Defined Areas of the Developing CNS in Zebrafish

The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes.

Nerve growth factor induces sphingomyelin accumulation in pheochromocytoma cells.

The pheochromocytoma cells are a well‐known model for studying the nerve growth factor (NGF)‐induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N‐lissamine rhodaminyl‐(12‐aminododecanoyl) D‐erythro‐sphingosine (C12‐LRh‐Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a 4‐fold increase of fluorescent SM content in PC12 cells, when loaded with C12‐LRh‐Cer. Treatment of PC12 cells with actinomycin D or cycloheximide completely abolishes the NGF‐induced elevation of SM. Inhibition of p140trkA receptor by AG‐879 prevents extracellular signal‐regulated kinase 1/2 phosphorylation and suppresses the increase of SM. Inhibition of protein kinase C (PKC), protein kinase A (PKA) and phosphatidylinositol 3‐kinase does not have any effect on NGF‐induced C12‐LRh‐SM accumulation. On the other hand, activation of PKA or PKC with simultaneous treatment with NGF has a synergistic effect on increase of SM content. The NGF‐induced SM increase in PC12 cells is an effect promoted by other differentiating agents like dibutyryl cyclic AMP or fibroblast growth factor‐2 but not by a mitogenic agent like epidermal growth factor.