Publications by authors named "Dario Brunetti"

19 Publications

  • Page 1 of 1

Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome.

Nat Commun 2021 03 26;12(1):1929. Epub 2021 Mar 26.

Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-22117-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997884PMC
March 2021

Therapeutic Approaches to Treat Mitochondrial Diseases: "One-Size-Fits-All" and "Precision Medicine" Strategies.

Pharmaceutics 2020 Nov 11;12(11). Epub 2020 Nov 11.

Medical Genetics and Neurogenetics Unit, Fondazione IRCCS Istituto Neurologico C. Besta, 20126 Milan, Italy.

Primary mitochondrial diseases (PMD) refer to a group of severe, often inherited genetic conditions due to mutations in the mitochondrial genome or in the nuclear genes encoding for proteins involved in oxidative phosphorylation (OXPHOS). The mutations hamper the last step of aerobic metabolism, affecting the primary source of cellular ATP synthesis. Mitochondrial diseases are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. The limited information of the natural history, the limitations of currently available preclinical models, coupled with the large variability of phenotypical presentations of PMD patients, have strongly penalized the development of effective therapies. However, new therapeutic strategies have been emerging, often with promising preclinical and clinical results. Here we review the state of the art on experimental treatments for mitochondrial diseases, presenting "one-size-fits-all" approaches and precision medicine strategies. Finally, we propose novel perspective therapeutic plans, either based on preclinical studies or currently used for other genetic or metabolic diseases that could be transferred to PMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pharmaceutics12111083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696526PMC
November 2020

Targeting Multiple Mitochondrial Processes by a Metabolic Modulator Prevents Sarcopenia and Cognitive Decline in SAMP8 Mice.

Front Pharmacol 2020 31;11:1171. Epub 2020 Jul 31.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

The age-dependent declines of skeletal muscle and cognitive functions often coexist in elderly subjects. The underlying pathophysiological mechanisms share common features of mitochondrial dysfunction, which plays a central role in the development of overt sarcopenia and/or dementia. Dietary supplementation with formulations of essential and branched-chain amino acids (EAA-BCAA) is a promising preventive strategy because it can preserve mitochondrial biogenesis and function. The senescence-accelerated mouse prone 8 (SAMP8) is considered an accurate model of age-related muscular and cognitive alterations. Hence, we aimed to investigate the progression of mitochondrial dysfunctions during muscular and cognitive aging of SAMP8 mice and to study the effects of a novel EAA-BCAA-based metabolic modulator on these changes. We evaluated body condition, motor endurance, and working memory of SAMP8 mice at 5, 9, 12, and 15 months of age. Parallel changes in protein levels of mitochondrial respiratory chain subunits, regulators of mitochondrial biogenesis and dynamics, and the antioxidant response, as well as respiratory complex activities, were measured in the quadriceps femoris and the hippocampus. The same variables were assessed in 12-month-old SAMP8 mice that had received dietary supplementation with the novel EAA-BCAA formulation, containing tricarboxylic acid cycle intermediates and co-factors (PD-0E7, 1.5 mg/kg/body weight/day in drinking water) for 3 months. Contrary to untreated mice, which had a significant molecular and phenotypic impairment, PD-0E7-treated mice showed preserved healthy body condition, muscle weight to body weight ratio, motor endurance, and working memory at 12 months of age. The PD-0E7 mixture increased the protein levels and the enzymatic activities of mitochondrial complex I, II, and IV and the expression of proliferator-activated receptor γ coactivator-1α, optic atrophy protein 1, and nuclear factor, erythroid 2 like 2 in muscles and hippocampi. The mitochondrial amyloid-β-degrading pitrilysin metallopeptidase 1 was upregulated, while amyloid precursor protein was reduced in the hippocampi of PD-0E7 treated mice. In conclusion, we show that a dietary supplement tailored to boost mitochondrial respiration preserves skeletal muscle and hippocampal mitochondrial quality control and health. When administered at the early onset of age-related physical and cognitive decline, this novel metabolic inducer counteracts the deleterious effects of precocious aging in both domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2020.01171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411305PMC
July 2020

Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer's disease-like pathology in human cerebral organoids.

Mol Psychiatry 2020 Jul 7. Epub 2020 Jul 7.

German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.

Mutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressing syndrome characterized by cerebellar ataxia, psychotic episodes, and obsessive behavior, as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1-knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPR) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid β in PITRM1-knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the other hand, over time, cerebral organoids generated from PITRM1-knockout iPSCs spontaneously developed pathological features of Alzheimer's disease (AD), including the accumulation of protein aggregates, tau pathology, and neuronal cell death. Single-cell RNA sequencing revealed a perturbation of mitochondrial function in all cell types in PITRM1-knockout cerebral organoids, whereas immune transcriptional signatures were substantially dysregulated in astrocytes. Importantly, we provide evidence of a protective role of UPR and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. Here, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPR that, in turn, modulates cytosolic quality control pathways. Thus, our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41380-020-0807-4DOI Listing
July 2020

Complete neural stem cell (NSC) neuronal differentiation requires a branched chain amino acids-induced persistent metabolic shift towards energy metabolism.

Pharmacol Res 2020 08 12;158:104863. Epub 2020 May 12.

Department of Diagnostics and Public Health, Section of Pharmacology, University of Verona, Verona, Italy. Electronic address:

Neural stem cell (NSC) neuronal differentiation requires a metabolic shift towards oxidative phosphorylation. We now show that a branched-chain amino acids-driven, persistent metabolic shift toward energy metabolism is required for full neuronal maturation. We increased energy metabolism of differentiating neurons derived both from murine NSCs and human induced pluripotent stem cells (iPSCs) by supplementing the cell culture medium with a mixture composed of branched-chain amino acids, essential amino acids, TCA cycle precursors and co-factors. We found that treated differentiating neuronal cells with enhanced energy metabolism increased: i) total dendritic length; ii) the mean number of branches and iii) the number and maturation of the dendritic spines. Furthermore, neuronal spines in treated neurons appeared more stable with stubby and mushroom phenotype and with increased expression of molecules involved in synapse formation. Treated neurons modified their mitochondrial dynamics increasing the mitochondrial fusion and, consistently with the increase of cellular ATP content, they activated cellular mTORC1 dependent p70S6 K1 anabolism. Global transcriptomic analysis further revealed that treated neurons induce Nrf2 mediated gene expression. This was correlated with a functional increase in the Reactive Oxygen Species (ROS) scavenging mechanisms. In conclusion, persistent branched-chain amino acids-driven metabolic shift toward energy metabolism enhanced neuronal differentiation and antioxidant defences. These findings offer new opportunities to pharmacologically modulate NSC neuronal differentiation and to develop effective strategies for treating neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.phrs.2020.104863DOI Listing
August 2020

Exploring the Relevance of Senotherapeutics for the Current SARS-CoV-2 Emergency and Similar Future Global Health Threats.

Cells 2020 04 8;9(4). Epub 2020 Apr 8.

Department of Translational Research, University of Pisa, 56126 Pisa, Italy.

The higher death rate caused by COVID-19 in older people, especially those with comorbidities, is a challenge for biomedical aging research. Here we explore the idea that an exacerbated inflammatory response, in particular that mediated by IL-6, may drive the deleterious consequences of the infection. Data shows that other RNA viruses, such as influenza virus, can display enhanced replication efficiency in senescent cells, suggesting that the accumulation of senescent cells with aging and age-related diseases may play a role in this phenomenon. However, at present, we are completely unaware of the response to SARS-CoV and SARS-COV-2 occurring in senescent cells. We deem that this is a priority area of research because it could lead to the development of several therapeutic strategies based on senotherapeutics or prevent unsuccessful attempts. Two of these senotherapeutics, azithromycin and ruxolitinib, are currently undergoing testing for their efficacy in treating COVID-19. The potential of these strategies is not only for ameliorating the consequences of the current emergence of SARS-CoV-2, but also for the future emergence of new viruses or mutated ones for which we are completely unprepared and for which no vaccines are available.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9040909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226793PMC
April 2020

A Special Amino-Acid Formula Tailored to Boosting Cell Respiration Prevents Mitochondrial Dysfunction and Oxidative Stress Caused by Doxorubicin in Mouse Cardiomyocytes.

Nutrients 2020 Jan 21;12(2). Epub 2020 Jan 21.

Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, 20129 Milan, Italy.

Anthracycline anticancer drugs, such as doxorubicin (DOX), can induce cardiotoxicity supposed to be related to mitochondrial damage. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem), supplemented with drinking water to middle-aged mice, was able to promote mitochondrial biogenesis in cardiac and skeletal muscle. To maximally favor and increase oxidative metabolism and mitochondrial function, here we tested a new original formula, composed of essential amino acids, tricarboxylic acid cycle precursors and co-factors (named 5), in HL-1 cardiomyocytes and mice treated with DOX. We measured mitochondrial biogenesis, oxidative stress, and BCAA catabolic pathway. Moreover, the molecular relevance of endothelial nitric oxide synthase (eNOS) and mechanistic/mammalian target of rapamycin complex 1 (mTORC1) was studied in both cardiac tissue and HL-1 cardiomyocytes. Finally, the role of Krüppel-like factor 15 (KLF15), a critical transcriptional regulator of BCAA oxidation and eNOS-mTORC1 signal, was investigated. Our results demonstrate that the 5 mixture prevents the DOX-dependent mitochondrial damage and oxidative stress better than the previous BCAAem, implying a KLF15/eNOS/mTORC1 signaling axis. These results could be relevant for the prevention of cardiotoxicity in the DOX-treated patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/nu12020282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071384PMC
January 2020

Mitochondrial peptidase loss-of-function in childhood cerebellar atrophy.

J Med Genet 2018 09 15;55(9):599-606. Epub 2018 May 15.

Department of Pediatrics, Medical Genetics Institute, Shaare Zedek Medical Center, Hebrew University-Hadassah School of Medicine, Jerusalem, Israel.

Objective: To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy.

Methods: Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast.

Results: Two brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 () c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same mutation on a shared haplotype. carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. is a mitochondrial matrix enzyme that degrades 10-65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids.

Conclusion: results in childhood-onset recessive cerebellar pathology. Severity of -related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jmedgenet-2018-105330DOI Listing
September 2018

Defective PITRM1 mitochondrial peptidase is associated with Aβ amyloidotic neurodegeneration.

EMBO Mol Med 2016 Mar;8(3):176-90

Department of Neurology, Haukeland University Hospital, Bergen, Norway Department of Clinical Medicine (K1), University of Bergen, Bergen, Norway

Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ-positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201505894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772954PMC
March 2016

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model.

Brain 2014 Jan 6;137(Pt 1):57-68. Epub 2013 Dec 6.

1 Unit of Molecular Neurogenetics, Foundation IRCCS Neurological Institute C. Besta, Milan, Italy.

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(-/-)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(-/-) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891449PMC
http://dx.doi.org/10.1093/brain/awt325DOI Listing
January 2014

Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

Hum Mol Genet 2012 Dec 13;21(24):5294-305. Epub 2012 Sep 13.

Unit of Molecular Neurogenetics, IRCCS Foundation Neurological Institute 'C. Besta', Milan, Italy.

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/dds380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510755PMC
December 2012

Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells.

J Cell Biochem 2013 Jan;114(1):134-43

Department of Comparative Biomedical Science, University of Teramo, 64100 Teramo, Italy.

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcb.24310DOI Listing
January 2013

C19orf12 and FA2H mutations are rare in Italian patients with neurodegeneration with brain iron accumulation.

Semin Pediatr Neurol 2012 Jun;19(2):75-81

Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the study of Mitochondrial Disorders in Children, IRCCS, Foundation Neurological Institute C. Besta, Milan, Italy.

Neurodegeneration with brain iron accumulation (NBIA) defines a wide spectrum of clinical entities characterized by iron accumulation in specific regions of the brain, predominantly in the basal ganglia. We evaluated the presence of FA2H and C19orf12 mutations in a cohort of 46 Italian patients with early onset NBIA, which were negative for mutations in the PANK2 and PLA2G6 genes. Follow-up molecular genetic and in vitro analyses were then performed. We did not find any mutations in the FA2H gene, although we identified 3 patients carrying novel mutations in the C19orf12 gene. The recent discovery of new genes responsible for NBIA extends the spectrum of the genetic investigation now available for these disorders and makes it possible to delineate a clearer clinical-genetic classification of different forms of this syndrome. A large fraction of patients still remain without a molecular genetics diagnosis, suggesting that additional NBIA genes are still to be discovered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.spen.2012.03.006DOI Listing
June 2012

Genetic engineering including superseding microinjection: new ways to make GM pigs.

Xenotransplantation 2010 Nov-Dec;17(6):397-410

Avantea, Laboratorio di Tecnologie della Riproduzione, Cremona, Italy.

Background: Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens.

Methods: A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons.

Results: The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task.

Conclusions: Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1399-3089.2010.00590.xDOI Listing
March 2011

Development, embryonic genome activity and mitochondrial characteristics of bovine-pig inter-family nuclear transfer embryos.

Reproduction 2010 Aug 7;140(2):273-85. Epub 2010 Jun 7.

Laboratorio di Tecnologie della Riproduzione, Avantea srl, Via Porcellasco 7/f, Cremona, Italy.

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine-pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16-25-cell stage for pig-bovine iSCNT embryos and at the four-cell stage for bovine-pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus-cytoplasm compatibility between these animal species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/REP-09-0578DOI Listing
August 2010

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.

Cloning Stem Cells 2008 Dec;10(4):409-19

Laboratorio di Tecnologie della Riproduzione, CIZ srl, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy.

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/clo.2008.0036DOI Listing
December 2008

Inverse relationship between scores on the quality of life questionnaire SF-12 and on the Aging Males' Symptoms scale in Italian men.

Aging Male 2008 Jun;11(2):77-82

Università di Milano, IRCGS Policlinico, Milan, Italy.

Objectives: To analyse the relation between results of the Aging Males' Symptoms (AMS) questionnaire for aging males, and of quality of life (QOL) questionnaire SF-12 and cardiovascular risk factors.

Methods: 1,927 men aged 55-85 years were interviewed by 56 general practitioners. During the interview the men were asked to fill in the AMS scale and the QOL questionnaire SF-12.

Results: Of 1,927 men 1,806 men filled correctly the AMS questionnaire. The mean SF-12 mental index was respectively 55.9 in men with a total AMS score indicating no impairment, 50.9 mild, 42.8 moderate, and 32.8 severe impairment. The corresponding values for the physical index were 51.2, 46.7, 40.8 and 32.3. A history of diabetes was associated with an increased risk of reporting moderate/severe impairment: in relation to the total AMS score the odds ratio, (OR), of moderate/severe impairment in comparison with no impairment was 1.6 (95%CI 1.2-2.1). A history of myocardial infarction and hypertension increased the risk (respectively OR 1.4 (95%CI 1.1-18) and 1.7 (95%CI 1.2-2.4)).

Conclusions: This study shows that higher AMS scores are associated with lower SF-12 indices and suggests that elevated values of the AMS score are associated with cardiovascular risk factors or diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/13685530701791262DOI Listing
June 2008

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.

Theriogenology 2007 Jan 1;67(1):90-8. Epub 2006 Nov 1.

Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy.

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.theriogenology.2006.09.011DOI Listing
January 2007

Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.

Stem Cells 2006 Nov 24;24(11):2514-21. Epub 2006 Aug 24.

Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ s.r.l., Cremona, Italy.

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organized into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organized neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here, we show that bovine inner cell masses from nuclear transfer and fertilized embryos, grown on feeders in serum-free medium, form polarized rosette structures expressing nestin, Pax6, Pax7, Sox1, and Otx2 and exhibiting interkinetic nuclear migration activity and cell junction distribution as in the developing neural tube. After in vitro expansion, neural rosettes give rise to p75-positive neural crest precursor cell lines capable of long-term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells, and chondrocytes, recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2, and Nkx6.1 and differentiation of mature astrocytes and neurons of central nervous system ventral identity, demonstrating appropriate response to inductive signals. All together, these findings indicate that neural rosettes directly derived from cloned and fertilized bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1634/stemcells.2006-0149DOI Listing
November 2006