Modelling Leigh Syndrome in pigs.

The generation of genetically modified animal models is a valuable tool to deepen scientific and medical knowledge of neurodegenerative diseases. Leigh syndrome associated with cytochrome C oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy characterized by neurodevelopmental regression and brain stem and basal ganglia lesions, frequently caused by mutations in the Surf1 gene, a nuclear gene that encodes a mitochondrial protein involved in COX assembly. The animal models of LS generated so far (Drosophila, Zebrafish, Mouse) fail to recapitulate simultaneously the human pathological and biochemical phenotype. In this thesis, we present the generation and the descriptive characterization of the first large animal model (swine) of Leigh Syndrome (LS), a well-characterized mitochondrial-derived ND. Since the complete and reliable sequence of swine Surf1 gene was not annotated in public databases, we sequenced the whole swine Surf1 gene to customize our genetic tools and precisely edit swine genome. To this purpose, we used the most recent genetic engineering technologies in terms of site-specific nucleases, as TALENs and CRISPR/Cas9, associated to a homologous recombination-based (HR) vector, to target swine Surf1 gene and generate an exon-specific double-strand break in order to knock out gene activity and thus reproduce null mutations reported in most human patients. Knockout (KO) and heterozygous animals were finally generated through Somatic Cell Nuclear Transfer (SCNT). A significant number of KO and heterozygous animals was generated and characterized, from both a clinical and a biochemical point of view. Surf1 KO animals exhibited an early lethal pathology onset, whereas heterozygous animals were completely comparable to wild type individuals. In conclusion, we successfully generated a Surf1 KO swine model, which needs to be further analysed as a promising tool to unveil the effective roles of Surf1 protein in metabolic pathways, in particular highlighting the perinatal period as a crucial moment

Cognitive profile in idiopathic autonomic failure: relation with white matter hyperintensities and neurofilament levels

To disclose the nature of cognitive deficits in a cohort of patients with idiopathic autonomic failure (IAF) by exploring the relation among cognitive functions, cardiovascular autonomic failure (AF) and clinical progression to another α-synucleinopathy (phenoconversion)

In vivo assessment of Lewy body and beta-amyloid copathologies in idiopathic normal pressure hydrocephalus: prevalence and associations with clinical features and surgery outcome

Background: Idiopathic normal pressure hydrocephalus (iNPH) is a clinico-radiological syndrome of elderly individuals likely sustained by different neurodegenerative changes as copathologies. Since iNPH is a potentially reversible condition, assessing neurodegenerative pathologies in vitam through CSF biomarkers and their influence on clinical features and surgical outcome represents crucial steps.Methods: We measured a-synuclein seeding activity related to Lewy body (LB) pathology by the real-time quaking-induced conversion assay (RT-QuIC) and Alzheimer disease core biomarkers (proteins total-tau, phospho-tau, and amyloid-beta) by immunoassays in the cerebrospinal fluid (CSF) of 293 iNPH patients from two independent cohorts. To compare the prevalence of LB copathology between iNPH participants and a control group representative of the general population, we searched for a-synuclein seeding activity in 89 age-matched individuals who died of Creutzfeldt-Jakob disease (CJD). Finally, in one of the iNPH cohorts, we also measured the CSF levels of neurofilament light chain protein (NfL) and evaluated the association between all CSF biomarkers, baseline clinical features, and surgery outcome at 6 months.Results: Sixty (20.5%) iNPH patients showed alpha-synuclein seeding activity with no significant difference between cohorts. In contrast, the prevalence observed in CJD was only 6.7% (p= 0.002). Overall, 24.0% of iNPH participants showed an amyloid-positive (A+) status, indicating a brain co-pathology related to A beta deposition. At baseline, in the Italian cohort, a-synuclein RT-QuIC positivity was associated with higher scores on axial and upper limb rigidity (p=0.003 and p =0.011, respectively) and lower MMSEc scores (p =0.003). A+ patients showed lower scores on the MMSEc (p =0.037) than A- patients. Higher NfL levels were also associated with lower scores on the MMSEc (rho = -0.213; p= 0.021). There were no significant associations between CSF biomarkers and surgical outcome at 6 months (i.e. responders defined by decrease of 1 point on the mRankin scale).Conclusions: Prevalent LB- and AD-related neurodegenerative pathologies affect a significant proportion of iNPH patients and contribute to cognitive decline (both) and motor impairment (only LB pathology) but do not significantly influence the surgical outcome at 6 months. Their effect on the clinical benefit after surgery over a more extended period remains to be determined

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices

Development of an in vitro test battery for the screening of the receptor-mediated mechanism and the spindle-poison mode of action of estrogenic compounds

The implementation of the REACH regulation has imposed the urgent need of developing alternative testing methods to screen large number of compounds more quickly and at lower costs. In this study, a battery of tests, suitable for reproductive toxicology testing, was developed with the objective of detecting the mechanism of action of estrogenic and xenoestrogenic compounds. With this aim, two compounds known for their estrogenic activity, diethylstilbestrol and 17ˇ-estradiol, were used to set up four differentin vitro tests: 1) bovine oocyte in vitro maturation assay, 2) bovine preimplantation embryo in vitro culture assay and 3) MCF-7 and 4) BALB/3T3 cell lines proliferation and cytotoxicity assay, respectively. The results show that this battery of tests allows to identify and to distinguish between two major mechanisms of action of (xeno)estrogenic compounds: the receptor-mediated mechanism and the spindle-poison effect on microtubules polimerization

The Applications of Genome Editing in Xenotransplantation

Higher living standards and better medical care are increasing the lifespan of people around the world. Aging populations, however, have an increased incidence of loss of function or failure of cell, tissue or organ. This has led to the development of new medical disciplines, such as organ transplantation and more recently regenerative medicine. Organ transplantation using human donors (allotransplantation) has made enormous progress thanks to the discovery of novel immunosuppressive drugs. However, the growing demand for organs far exceeds the number of organs potentially available from human donors. Xenotransplantation, namely transplantation between animal donors and man, offers the opportunity to use healthy and highly specialized cells, tissues or solid organs readily available for immediate transfer to patients requiring replacement therapy (Ekser et al., 2012). The therapeutic potential of xenotransplantation is wide, some already in clinical use like bioprosthetic heart valves, decellularized pig tissues (skin, ligaments, bone and cartilage), polyclonal antisera, and pancreatic islet, or in a pre-clinical phase like kidney, heart, liver, lung, cornea, and dopaminergic neurons. The pig is a very suitable species for xenotransplantation for reasons that are well documented in the literature, including physiological and anatomical features, and the availability of a high resolution map of the genome. Moreover, the use of pigs for clinical purposes raises little concern from the wider public, because they are already bred by the millions for meat production worldwide. At present, the use of bioprosthetic heart valves of animal origin is a well established xenotransplantation procedure in clinical practice; however, pig islet xenotransplantation has just entered clinical trials (http://www.lctglobal.com/products/ diabecell/about-type-1-diabetes), and life supporting solid organs transplanted into nonhuman primates still do not survive long enough to warrant implementation of clinical trials (Le Bas-Bernardet et al., 2011) although heterotopic heart transplantation in a primate model has resulted in the remarkable survival of almost three years (Mohiuddin et al., 2015). Therefore, several issues still need to be addressed from the safety point of view, and a number of immunological hurdles have been identified (Table 1) and are currently being addressed at multiple levels (Griesemer et al., 2014). It is expected that the development of novel immunosuppressive strategies for allotransplantation and xenotransplantation, the modification of the immunogenicity of the donor pig through genetic engineering and, possibly the induction of immune tolerance, a phenomenon occasionally observed in allotransplantation, will all contribute to bringing xenotransplantation closer to the clinic (Ekser et al., 2012)

Synthetic Thyroid Hormone Receptor-β Agonists Promote Oligodendrocyte Precursor Cell Differentiation in the Presence of Inflammatory Challenges

Oligodendrocytes and their precursors are the cells responsible for developmental myelination and myelin repair during adulthood. Their differentiation and maturation processes are regulated by a complex molecular machinery driven mainly by triiodothyronine (T3), the genomic active form of thyroid hormone, which binds to thyroid hormone receptors (TRs), regulating the expression of target genes. Different molecular tools have been developed to mimic T3 action in an attempt to overcome the myelin repair deficit that underlies various central nervous system pathologies. In this study, we used a well-established in vitro model of neural stem cell-derived oligodendrocyte precursor cells (OPCs) to test the effects of two compounds: the TRβ1 ligand IS25 and its pro-drug TG68. We showed that treatment with TG68 induces OPC differentiation/maturation as well as both the natural ligand and the best-known TRβ1 synthetic ligand, GC-1. We then described that, unlike T3, TG68 can fully overcome the cytokine-mediated oligodendrocyte differentiation block. In conclusion, we showed the ability of a new synthetic compound to stimulate OPC differentiation and overcome inflammation-mediated pathological conditions. Further studies will clarify whether the compound acts as a pro-drug to produce the TRβ1 ligand IS25 or if its action is mediated by secondary mechanisms such as AMPK activation