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Aristaless-related homeobox (ARX) is a crucial transcription factor involved in several interneuronopathies, including infantile spasms (ISSX1) and West Syndrome. Its polyAlanine motifs are hot spots for elongations, frequently found in kids affected by malignant Epilepsy with limited therapeutic options. Although in the last years several medical therapies have been tested to prevent epilepsy in West syndrome patients, an effective treatment is still needed. Noteworthy, the early onset of infantile spams followed by developmental delay and poor seizure control make these patients at high risk for suddenly unexpected death (SUDEP). The overall objectives of our project were to study Aristaless-related Homeobox Epilepsy models with the final goal of identifying druggable target molecules.

Studying molecular convergence in neurodevelopmental disorders caused by mutations in specific disease-related genes permits us to define druggable molecular pathways. The purpose of our study is to assess the degree of damage associated with the ARX-KDM5C and to establish a correlation between similar phenotypes and same cellular functions. Mutations in Aristaless-related homeobox gene (ARX), a homeotic transcription factor with a key role in interneuron maturation, have been found in a spectrum of X-chromosome phenotypes including cortical malformations, chronic Epilepsy and X-Linked Intellectual Disabilities (XLID). About Lysine-specific demethylase 5C (KDM5C), its mutations have been reported as an important cause of XLID. Its protein is a histone demethylase acting as transcriptional repressor during brain development. Here we summarize functional analysis of two classes of ARX mutations : 1. PolyAlanine elongations affecting the first and the second PolyA tracts, frequently found in patients with Epilepsy or XLID; and 2. Missense mutations clustered in the paired-type homeodomain (HD) found in patients with Lissencephaly with abnormal genitalia (XLAG). Thus, we propose a "fault disease model" showing that the degree of spectrum of defects in transcription of ARX-target genes correlate with the severity of the neurophenotypes associated with ARX mutations.

ARX (Aristaless-related homeobox gene), is a crucial gene for development of interneuron in the fetal brain, implicated in intellectual disability associated with a severe form of Epilepsy. It is caracterized by a trinucleotide (GCN) repeats with high tendency to expand causing the alanine elongations (polyA). We report here an innovative long non-coding RNA-based technology, the application of the SINEUP-strategy, to correct ARX deficiency. These method is able to enhance protein translation by using synthetic long non coding RNA (lncRNA). SINEUPs are a new class of RNA therapeutics that allows to design gene-specific approaches to treat, malignant epilepsy diseases and many other neuropathologies with seizure linked to insufficient activity of chromatin and/or transcriptional regulators.

The X-linked ARX gene encodes the Aristaless-related homeobox protein, ARX, an interneuron-specific transcription factor (TF) with a key role in GABAergic interneuron migration and maturation. Expansions of the first two polyalanine tracts (PA1 and PA2) cause refractory epilepsy (RE) with infantile spasms or X-linked Intellectual Disability (XLID). Here we report on an evolutionary conserved druggable ARX-dependent pathway damaged by the faulty functioning of the polyalanine elongated TFs, exerted through a poorly-understood pathogenic mechanism. We have combined the molecular validation of the conserved interaction of ARX with KDM5C from mammals to C.elegans with the analysis of relative neuronal outcomes in order to explore potential compounds able to correct molecular faults behind the ARX polyalanine dysfunction.

The X-linked ARX gene encodes the Aristaless-related homeobox protein, which belongs to a subset of morphogenetic transcription factors with a crucial role in cerebral development and patterning. Mutations in ARX cause a wide spectrum of X-linked neurodevelopmental disorders including lissencephaly, a severe cortical malformation and a catastrophic epileptic encephalopathy with recurrent and resistant seizures. The health problem to find efficient treatments for ARX-related diseases highlights the need for understanding the components and pathways that regulate brain development. Because the neuronal functioning is maintained by a complex regulatory network, the use of powerful genetic models like mouse and worm can complement studies on human genetics and physiology by offering new opportunities to dissect complicated and evolutionary conserved regulatory circuits. Here we describe for the first time the conservation of an ARX-dependent disease-pathway among human, mouse and worm establishing a gene-phenotype association from one organism to another.

Mistakes in histone methylation-demethylation rounds have been directly involved in several forms of Intellectual Disability (ID) with Epilepsy and/or Refractory Epilepsy (RE). Lysine-specific demethylase 5C (KDM5C) is an X-linked gene, which encodes a chromatin JmjC eraser with H3K4me2/3 demethylase activity. KDM5C is frequently mutated in a spectrum of X-linked ID (XLID) and/or RE. It functions as a transcriptional repressor that is critical for transition of neural progenitors to neurons. We identified a disease path, linking functionally KDM5C to another XLID/Epilepsy gene, encoding the homeotic transcription factor ARX, whose mutations impair severely KDM5C transcript regulation.Ongoing efforts will allow us to identify druggable hallmarks that could open up towards the exploitation of potential strategies to treat the growing group of ID and RE diseases caused by defects in chromatin and/or transcriptional regulators.

Reproduction in lizards, as in other vertebrates, is regulated by a complex interplay of ambient and endogenous factors. Changes in temperature and photoperiod in particular modulate the release of gonadotropin and ovarian factors and these, directly or indirectly, control different ovarian activities. In this review, mechanisms regulating oocyte recruitment and selection will be discussed and integrated with original data on the Italian wall lizard Podarcis sicula. In particular, starting from the colonization of the gonadal anlage by the primordial germ cells we will show how oocytes and follicles recruitment proceed throughout the year and how these two processes are influenced by exogenous FSH, oestradiol and progesterone in different periods of the ovarian cycle. In addition, by reporting results of unilateral ovariectomy (ULO), follicle ablation (ULA) and follicle puncture (PU) experiments we will demonstrate that clutch size in this species is probably regulated by two ovarian factors, one inhibiting and the other promoting the recruitment of new primary follicles from the pool of prefollicular oocytes. We hypothesize the former factor being released systemically by the oocyte to stop follicle recruitment by a dose dependent mechanism. The latter factor would be produced by the follicle cells and would have a promoting effect by acting at a strictly local level. This control mechanism cannot be bypassed by administering gonadotropins or steroids, by placing the animals at high temperature or by exposure to cadmium, a known endocrine disrupter that promotes prefollicular oocyte recruitment.

Mis-steps in histone methylation-demethylation rounds have been directly involved in several forms of Intellectual Disability (ID) with Epilepsy. Lysine-specific demethylase 5C (KDM5C) is an X-linked gene, which encodes a chromatin JmjC eraser with H3K4me2/3 demethylase activity. KDM5C is frequently mutated in a spectrum of XLID and/or malignant Epilepsy. It functions as a transcriptional repressor and interacts with REST/NSRF, a master epigenetic hub that is critical for transition of neural progenitors to neurons.Because chromatin modifications are reversible, it is possible that epigenetic drugs could compensate for the KDM5C-H3K4me3 deregulation. To achieve this aim, we screened a number of compounds targeting chromatin enzymes. We used, as cell disease model, neuronally-differentiated Arx KO/Kdm5C-depleted ES cells, which show GABAergic abnormalities in association with a global increase of H3K4me3 signal. Ongoing efforts in this direction allowed us to identify druggable hallmarks that could open up towards the exploitation of potential strategies to treat the growing group of ID and Epilepsy diseases that are caused by defects in chromatin and/or transcriptional regulators.

Mis-steps in histone methylation-demethylation rounds have been directly involved in several forms of Intellectual Disability (ID) with Epilepsy. Lysine-specific demethylase 5C (KDM5C) is an X-linked gene which encodes a chromatin JmjC eraser with H3K4me2/3 demethylase activity. KDM5C is frequently mutated in a spectrum of XLID and/or malignant Epilepsy. Noteworthy, a defective KDM5C-H3K4me3 path has been found in association with a mis-regulation of XLID/Epilepsy effector genes. We report here on methods to correct KDM5C fault by testing epi-drugs to target KDM5C-H3K4me3 axis, specifically.In particular, we have undertaken an in vitro analysis of a number of compounds targeting chromatin enzymes with the aim of modifying the accessibility of the transcription machinery to KDM5C. A strong up-regulation of Kdm5C/KDM5C has been obtained in presence of an inhibitor of HDAC at different points of in vitro differentiation, both in WT and Kdm5C-defective cells. these methods allows to design new approaches to treat ID, malignant epilepsy diseases and many other neuropathologies with seizure linked to insufficient activity of chromatin and/or transcriptional regulators.

EFFETTI DEL METALLO SULLA STRUTTURA ED ORGANIZZAZIONE DEL FOLLICOLO DELLA LUCERTOLA.

Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, -aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.

caratterizzazione del contenuto in glucidi degli alveoli corticali

Polyalanine expansion mutations of Aristaless-related homeobox gene (ARX) cause a spectrum of X-chromosome phenotypes with Intellectual Disabilily (ID) and various forms of malignant paediatric epilepsy. We have established that ARX regulates and binds a CNE element in the 5' region of the lysine (K)-specific demethylase 5C (KDM5C), a known XLID gene involved in chromatin remodeling and neuronal maturation. By in vitro studies, we have analyzed five Polyalanine mutants establishing a decreased trans-activating activity and a reduced, but not abolished, binding to the KDM5C regulatory region. By quantitative RT-PCR, we have showed in murine Arx-KO ES cells and neurospheres a dramatic downregulation of the Kdm5C mRNA levels that leads to a decrease of the KDM5C protein. In Arx KO GABA-oriented model, which presents severe abnormalities in dendrite formation and GABAergic maturation, we have found a KDM5C reduction in coupling with a global increase of H3K4me3 signalling, potentially due to a compromised KDM5C activity. Since H3K4me3 is the hallmark of open chromatin, ARX-dependent KDM5C defects could compromise cyclical rounds of methylation-demethylation and consequently the chromatin remodelling. Starting from these data, we are now able to break up a unique ARX-dependent epigenetic road involved in epileptogenesis, suggesting that the molecular pathogenesis of ARX Polyalanine mutations may be in part caused by aberrant histone demethylation as a result of KDM5C defect. As chromatin modifications are reversible, it is possible that epigenetic drugs could compensate KDM5C-H3K4me3 deregulation opening further studies to cure or ameliorate ARX Polyalanine-related epilepsy.

Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metabolic pathway is described to be crucial in determining ES cell fate.

The exposure to environmental toxicants such cadmium (Cd) is an important research area in wildlife protection. In this study, the effect of Cd oral administration on the ovarian structure and function and on reproductive performance of the Italian wall lizard Podarcis sicula was studied. In vivo, adult female lizards were randomly assigned to three groups. Cd was given with food in single dose and in multiple doses 3 days/week for 4 weeks at dose of 1.0 μg/g body weight. Following euthanasia, the ovaries were removed and analyzed for morpho-functional changes. Results demonstrated that Cd increases prefollicular germ cells number; the evaluation of the number of follicles detects significantly higher number of atretic growing follicles, whereas primary follicles remain unchanged with respect to controls. After Cd treatments, follicles are deformed by the presence of large protrusions and a general dysregulation in the follicle organization is observed. The zona pellucida is also affected. Cd causes alteration in sugar metabolism and in metallothionein gene expression. Finally, Cd administration significantly reduces clutch size and dramatically increases embryo mortality. In conclusion, data here described show that Cd induces morpho-functional alterations in lizard follicles and indicates that these are responsible for a significant impairment of oogenesis. The effects of the dose are time independent, persisting essentially unchanged regardless of single or multiple administration, so it can be concluded that even occasional, sublethal Cd contamination may significantly impair reproductive performance in these animals.

In physiologic concentrations, endogenous reactive oxygen species (ROS) help to maintain homeostasis. However, when ROS accumulate in excess for prolonged periods of time, they cause chronic oxidative stress and adverse effects. Oxidative stress is linked to the pathogenesis and pathobiochemistry of various diseases, including cancer, diabetes, cardiovascular and neurodegeneration. Organisms have evolved extensive enzymatic defense systems to protect against the deleterious effects of oxidants. The tripeptide glutathione (GSH) is part of these antioxidant systems that protect cells and tissues from oxidative damage. GSH is believed to function as an important cellular redox buffer and has been suggested to be involved in determining cell fate decisions, such as proliferation, differentiation and apoptosis. Mouse genetic models of disease are often limited by the inherent variability of animal experiments, the limited mouse life span, and the difficulties in manipulating whole animals. For instance, genetic rescue experiments and toxicological dose-response studies are impractical in whole animals. Furthermore, genetic cell models are more readily amenable to molecular dissection of disease mechanisms than are whole animals. Primary cultures or established cell lines are commonly used to analyse the cytotoxic effect of chemical factors, drugs and xenobiotics in vitro. An alternative approach will be provided by permanent lines of pluripotent embryonic stem (ES) cells, which are able to differentiate into specialized somatic cell types in vitro. ES cells carrying genetic mutations are a suitable system to study events occurring during development. In fact, during ES cell differentiation, tissue-specific genes, proteins, as well as functional properties are expressed in a developmentally regulated manner recapitulating processes of early embryonic development. In this review we report data showing the use of genetically modified ES-derived neurons, erythrocytes and cardiomyocytes in studying the role of oxidative stress in cell death and differentiation.