FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba

International audienceBACKGROUND: Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes

Generation of pre-implantation pig SCNT embryos harboring the amyotrophic lateral sclerosis related hSOD1G93A gene

Introduction. ALS is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. Pathogenic hypotheses mainly rely on transgenic rodent models however doubts have been raised about their suitability to faithfully reproduce the human disease. Therefore, a more suitable model is strongly demanded to provide a better tool to study the disease and to facilitate the preclinical findings extrapolation. Swine model plays an emerging role in biomedical research as its anatomical, physiological and biochemical features are more closely related to human species. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic swine blastocysts carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces the ALS patients phenotype progression. Material and Methods. Using the Multisite Gateway System (Invitrogen) we obtained the \u201cEntryClone\u201d pENTRL1L2- hSODG93A, containing the cDNA coding the hSOD1G93A gene whose open reading frame was confirmed by sequencing, and the \u201cDestinationVector\u201d pMGOrfA5\ua23\u2019MARpuro5171. The exchange reaction between the \u201cDestinationVector\u201d and the \u201cEntryClone\u201d was mediated by LR Clonase-Invitrogen, and was used to transform chemically competent E.coli cells (OneShotMatch1-Invitrogen). The resulting \u201cExpressionVector\u201d pMG5\ua23\u2019MARPuro-hSODG93A was purified and analyzed. The same construct was successfully used to develop an ubiquitous EGFP expression vector that maintains high expression level through the next generation of pigs. This vector carries the pCAGGS hybrid promoter (CMV-IE enhancer + chicken \u3b2-actin promoter) inserted between two insulators (5\u2032MAR of chicken lysozyme gene) to prevent positional or copy number silencing effects. 5 \ub5g of linearized vector were used to transfect 1 7 106 Pig Adult Fibroblast (PAF) cultured in DMEM/ M199[1:1] + 10%FCS+1% \u3b2-FGF by Nucleofector (Amaxa Biosystem). Transgene expression was detected by ICC using an anti-hSOD1 antibody ([1:200]07\u2013403 Millipore) to select PAF colonies to be employed as donor cells. The endogenous expression level was evaluated in Huvec cells. SCNT-embryos were reconstructed following a zona-free method, as described previously. Transgenic hSOD1G93A-embryos were finally grown in vitro to assess morphology, development and survival. Results. PAFs showed a transgene expression level ranging from lower (about 0.5 times) to higher (about 3\u20134 times) than the endogenous one. Thirteen hSOD1G93A-PAF colonies carrying the complete range of expression levels were employed in eight SCNT experiments. A total of 1,630 oocytes were enucleated and 1430 SCNT-embryos were obtained. The average viable embryos percentage was 39.16% of the total manipulated oocytes. These results, consistent with those obtained from similar experiments conducted with other transgenes, have ruled out the hSOD1G93A lethality in the pre-implantation embryonic phases, regardless of its expression level. Conclusion. On the basis of these results the hSOD1G93Ablastocysts can be generated by SCNT. The next step will be to implant them in recipient sows to generate offspring. Taking into account the similarity in size and physiology of neuromuscolar system, a swine ALS model may represent a more suitable model in reproducing the human disease than current transgenic rodents

Amyotrophic lateral sclerosis (ALS) swine models: Production and preliminary characterization

Introduction. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. As employment of transgenic SOD1 rodent models in ALS research didn’t result in an improvement of patient prognosis, another model, more closely related to human species, is strongly demanded by the scientific community. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic blastocysts and swine carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces patients phenotype progression. Materials and Methods. SCNT blastocysts on fifth day of development were transplanted by midventral laparatomy to the synchronized sows uterus. A cesarean delivery was performed at the 114th day of gestation. In order to achieve a preliminary characterization of our swine model, tissue banking was performed on stillborn piglets and on animals that died soon after birth. Immunocytochemistry on ear biopsy fibroblasts and western blot on homogenized snap-shot frozen tissues (rabbit policlonal antibody 07–403 Millipore, concentration 1:200 and 1:1000 respectively) were performed. To assess SOD1G93A deposition pattern Immunohistochemistry (rabbit policlonal antibody GTX 100659; 1:250) and Immunofluorescence (GTX 100659; 1:250 and NeuN MAB377; 1:1000) were employed on FFPE tissues. Genomic SOD1G93A swine DNA digested by SalI+BglII (10 U/μg DNA) was hybridized with SOD-DIG probes (20 ng/ml) to assess transgene integrations number by Southern Blot. Results and Conclusions. The transfer of 638 embryos to eight recipient sows resulted in four pregnancies and in the birth of 16 vital and 12 stillborn piglets (mean blastocyst development to term efficacy, 8.78%). Five animals developed normally while the remaining piglets died due to events commonly reported in commercial herds. The transgenic protein expression was confirmed by both immunocytochemistry and western blot. Furthermore Southern blot revealed a transgene integration number ranging from 1 to about 6 copies. IHC demonstrated granular mutant protein aggregates in both perikarya and neurites of neurons (nucleus labeled with NeuN in immunofluorescence experiment) in brain (from area hypothalamica lateralis to the third ventricle) and in spinal cord neurites. Despite these encouraging results, further molecular and pathological investigations are required since data have been obtained in stillborn or extremely young animals. A detailed phenotypical characterization, adapting to pig currently employed human diagnostic devices, is in progress on adult living swin