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Supplementary Figures 1-4, Table 1 from Novel Dyskerin-Mediated Mechanism of p53 Inactivation through Defective mRNA Translation

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)–containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors. Cancer Res; 70(11); 4767–77. ©2010 AACR.

According to established cognitive neuroscience knowledge based on studies on disabled and typically developing readers, reading is based on a dual-stream model in which a phonological-dorsal stream (left temporo-parietal and inferior frontal areas) processes unfamiliar words and pseudowords, whereas an orthographic-ventral stream (left occipito-temporal and inferior frontal areas) processes known words. However, correlational neuroimaging, causal longitudinal, training, and pharmacological studies have suggested the critical role of visuo-spatial attention in reading development. In a double blind, crossover within-subjects experiment, we manipulated the neuromodulatory effect of a short-term bilateral stimulation of posterior parietal cortex (PPC) by using active and sham tRNS during reading tasks in a large sample of young adults. In contrast to the dual-stream model predicting either no effect or a selective effect on the stimulated phonological-dorsal stream (as well as to a general multisensory effect on both reading streams), we found that only word-reading performance improved after active bilateral PPC tRNS. These findings demonstrate a direct neural connectivity between the PPC, controlling visuo-spatial attention, and the ventral stream for visual word recognition. These results support a neurobiological model of reading where performance of the orthographic-ventral stream is boosted by an efficient deployment of visuo-spatial attention from bilateral PPC stimulation.

Although developmental reading disorders (developmental dyslexia) have been mainly associated with auditory-phonological deficits, recent longitudinal and training studies have shown a possible causal role of visuo-attentional skills in reading acquisition. Indeed, visuo-attentional mechanisms could be involved in the orthographic processing of the letter string and the graphemic parsing that precede the grapheme-to-phoneme mapping. Here, we used a simple paper-and-pencil task composed of three labyrinths to measure visuo-spatial attention in a large sample of primary school children (n = 398). In comparison to visual search tasks requiring visual working memory, our labyrinth task mainly measures distributed and focused visuo-spatial attention, also controlling for sensorimotor learning. Compared to typical readers (n = 340), children with reading difficulties (n = 58) showed clear visuo-spatial attention impairments that appear not linked to motor coordination and procedural learning skills implicated in this paper and pencil task. Since visual attention is dysfunctional in about 40% of the children with reading difficulties, an efficient reading remediation program should integrate both auditory-phonological and visuo-attentional interventions.

Human perception of a visual scene is hierarchically organized. Such rapid, albeit coarse, global processing allows people to create a useful context in which local details can be successively allocated. Lack of the typical hierarchical global-to-local visual processing is longitudinally predictive of future reading difficulties in pre-readers, which suggests that an atypical local perception can interfere with reading skill acquisition. Global and local Navon tasks were used to induce a transient perceptual priming before a reading-aloud task. We tested the effect of an atypical local perception on lexical and sublexical reading routes in typical adult readers. Local (vs. global) priming resulted in a slower phonological access to irregular, relative to regular, words. By contrast, pseudoword reading was not affected by local (vs. global) perceptual priming. Our findings demonstrate that, in typical adult readers, local priming impairs the fast processing of the letter string useful for lexical reading.

For about 10% of children reading acquisition is extremely difficult because they are affected by a heritable neurobiological disorder called developmental dyslexia (DD), mainly associated to an auditory-phonological disorder. Visual crowding is a universal phenomenon that impairs the recognition of stimuli in clutter, such as a letter in a word or a word in a text. Several studies have shown an excessive crowding in individuals with DD, but the causal link between excessive crowding and DD is not yet clearly established. An excessive crowding might be, indeed, a simple effect of DD due to reduced reading experience. The results of five experiments in 181 children reveal that: (i) an excessive crowding only at unattended locations characterizes an unselected group of children with DD (Experiment 1); (ii) an extra-large spaced text increases reading accuracy by reducing crowding in an unselected group of children with DD (Experiment 2); (iii) efficient attentional action video game trainings reduce crowding and accelerate reading speed in two unselected groups of children with DD (Experiment 3 and 4), and; (iv) pre-reading crowding longitudinally predicts future poor readers (Experiment 5). Our results show multiple causal links between visual crowding and learning to read. These findings provide new insights for a more efficient remediation and prevention for DD.

Learning to read is extremely difficult for about 10% of the children because they are affected by a heritable neurobiological disorder called developmental dyslexia (DD). The causal role of cognitive deficits typically associated to DD can be investigated through intervention studies. It has been demonstrated that visual-attention and reading speed could be simultaneously improved by using action video game (AVG) training both in shallow and deep alphabetic orthographies. Here, in a clinical study we showed that after this general-domain behavioral intervention both the phonological decoding speed and phonological short-term memory were increased only in DD children in which their video game score was improved. These findings confirm that an AVG training enhances the efficiency of both visual and auditory processing. The plasticity of the multi-sensory attentional network could explain the reading and reading-related improvements induced by the AVG training in children with DD.

Reading acquisition is extremely difficult for about 5% of children because they are affected by a heritable neurobiological disorder called developmental dyslexia (DD). Intervention studies can be used to investigate the causal role of neurocognitive deficits in DD. Recently, it has been proposed that action video games (AVGs)-enhancing attentional control-could improve perception and working memory as well as reading skills. In a partial crossover intervention study, we investigated the effect of AVG and non-AVG training on attentional control using a conjunction visual search task in children with DD. We also measured the non-alphanumeric rapid automatized naming (RAN), phonological decoding and word reading before and after AVG and non-AVG training. After both video game training sessions no effect was found in non-alphanumeric RAN and in word reading performance. However, after only 12 h of AVG training the attentional control was improved (i.e., the set-size slopes were flatter in visual search) and phonological decoding speed was accelerated. Crucially, attentional control and phonological decoding speed were increased only in DD children whose video game score was highly efficient after the AVG training. We demonstrated that only an efficient AVG training induces a plasticity of the fronto-parietal attentional control linked to a selective phonological decoding improvement in children with DD. Special Issue: Multiple Neurocognitive Deficits and Dyslexia ispartof: Brain Sciences vol:11 issue:2 pages:1-18 ispartof: location:Switzerland status: published

Il disturbo da comportamento dirompente e uno dei piu frequenti motivi di consultazione neuropsichiatrica presso i servizi ambulatoriali per l’eta evolutiva in Italia (Frigerio et al., 2006), cosi come in altri paesi (Steiner e Remsing, 2007). Tale disturbo, comprendente il disturbo oppositivo provocatorio e il disturbo della condotta, e contraddistinto da un gruppo eterogeneo di pazienti con una vasta gamma di problemi comportamentali, che variano da un relativamente minore atteggiamento di sfida e scatti d’ira a piu serie violazioni, quali aggressione fisica, distruttivita e furto (American Psychiatric Association, 2000). I problemi maggiori ai quali rischiano di andare incontro i bambini con disturbo oppositivo provocatorio e disturbo della condotta sono principalmente: rifiuto dei pari, fallimento scolastico, abuso di sostanze, e criminalita (Burke, Arkowitz e Dunn, 2002; Odgers et al., 2008), con conseguenti costi sociali elevati (Kolko et al., 2009). La persistenza di tali problematiche comportamentali nei bambini e il loro ruolo nello sviluppo di rilevanti problemi clinici, mette in luce il bisogno di ulteriori studi nel campo dei fattori di rischio del disturbo da comportamento dirompente (Martens, 2000; Simonoff et al., 2004).

Dyslexia is a neurodevelopmental disorder with an atypical activation of posterior left-hemisphere brain reading networks (i.e., temporo-occipital and temporo-parietal regions) and multiple neuropsychological deficits. Transcranial direct current stimulation (tDCS) is a tool for manipulating neural activity and, in turn, neurocognitive processes. While studies have demonstrated the significant effects of tDCS on reading, neurocognitive changes beyond reading modulation have been poorly investigated. The present study aimed at examining whether tDCS on temporo-parietal regions affected not only reading, but also phonological skills, visuo-spatial working memory, visuo-spatial attention, and motion perception in a polarity-dependent way. In a within-subjects design, ten children and adolescents with dyslexia performed reading and neuropsychological tasks after 20 min of exposure to Left Anodal/Right Cathodal (LA/RC) and Right Anodal/Left Cathodal (RA/LC) tDCS. LA/RC tDCS compared to RA/LC tDCS improved text accuracy, word recognition speed, motion perception, and modified attentional focusing in our group of children and adolescents with dyslexia. Changes in text reading accuracy and word recognition speed—after LA/RC tDCS compared to RA/LC—were related to changes in motion perception and in visuo-spatial working memory, respectively. Our findings demonstrated that reading and domain-general neurocognitive functions in a group of children and adolescents with dyslexia change following tDCS and that they are polarity-dependent.

Visual crowding is the difficulty in perceiving a target in the presence of nearby flankers. Most neurophysiological studies of crowding employed functional neuroimaging, but because of its low temporal resolution, no definitive answer can be given to the question: is crowding arising at the earliest or at later stages of visual processing? Here, we used a classic letters crowding paradigm in combination with electroencephalography (EEG). We manipulated the critical space between peripheral target and flankers, while ensuring a proper control of basic stimulus characteristics. Analyses were focused on event-related potentials (ERPs) and oscillatory activity in the alpha (8-12 Hz), beta (15-30 Hz) and gamma (30-80 Hz) bands. At the ERP level, we found that the first sign of a crowding-induced modulation of EEG activity was a suppression of the N1 component. Oscillatory analysis revealed an early stimulus-evoked gamma enhancement and a later alpha reduction that, however, were not influenced by the amount of crowding. Importantly, reduction in the beta band reflected the amount of crowding (i.e., stronger reduction for strong relative to mid crowding condition) and correlated with individual behavioral performance. Collectively, these findings show that crowding for complex objects emerges at later stages of visual processing, possibly as a result of large-scale network interaction.

The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.

Cancer stem cells (CSCs) are affected by the local micro-environment, the niche, in which inflammatory stimuli and hypoxia act as steering factors. Here, two nuclear receptors (NRs) agonists, i.e. pioglitazone (PGZ), a ligand of peroxisome proliferator activated receptor-γ, and 6-OH-11-O-hydroxyphenanthrene (IIF), a ligand of retinoid X receptors, were investigated for their capability to interference with the cross-talk between breast CSCs and the niche compartment. We found that IIF potentiates the ability of PGZ to hamper the mammospheres-forming capability of human breast tumours and MCF7 cancer cells, reducing the expression of CSCs regulatory genes (Notch3, Jagged1, SLUG, Interleukin-6, Apolipoprotein E, Hypoxia inducible factor-1α and Carbonic anhydrase IX). Notably, these effects are not observed in normal-MS obtained from human breast tissue. Importantly, NRs agonists abolish the capability of hypoxic MCF7 derived exosomes to induce a pro-inflammatory phenotype in mammary glands fibroblasts. Moreover, NRs agonist also directly acts on breast tumour associated fibroblasts to downregulate nuclear factor-κB pathway and metalloproteinases (MMP2 and MMP9) expression and activity. In conclusion, NRs agonists disrupt the inflammatory cross-talk of the hypoxic breast CSCs niche.

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)–containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors. Cancer Res; 70(11); 4767–77. ©2010 AACR.

Cancer stem cells (CSCs) are affected by the local micro-environment, the niche, in which inflammatory stimuli and hypoxia act as steering factors. Here, two nuclear receptors (NRs) agonists, i.e. pioglitazone (PGZ), a ligand of peroxisome proliferator activated receptor-γ, and 6-OH-11-O-hydroxyphenanthrene (IIF), a ligand of retinoid X receptors, were investigated for their capability to interference with the cross-talk between breast CSCs and the niche compartment. We found that IIF potentiates the ability of PGZ to hamper the mammospheres-forming capability of human breast tumours and MCF7 cancer cells, reducing the expression of CSCs regulatory genes (Notch3, Jagged1, SLUG, Interleukin-6, Apolipoprotein E, Hypoxia inducible factor-1α and Carbonic anhydrase IX). Notably, these effects are not observed in normal-MS obtained from human breast tissue. Importantly, NRs agonists abolish the capability of hypoxic MCF7 derived exosomes to induce a pro-inflammatory phenotype in mammary glands fibroblasts. Moreover, NRs agonist also directly acts on breast tumour associated fibroblasts to downregulate nuclear factor-κB pathway and metalloproteinases (MMP2 and MMP9) expression and activity. In conclusion, NRs agonists disrupt the inflammatory cross-talk of the hypoxic breast CSCs niche.

For children affected by developmental dyslexia learning to read is extremely difficult. Pre-reading visual attention predicts future reading acquisition skills. Action video game (AVG) training increases attentional functioning and induces learning that transfers well beyond the task domain, such as reading. We investigated the effects of AVG training on predictors of future reading acquisition (i.e., visuo-spatial attention, auditory-phonological processing and rapid naming skills) and on the dense-array EEG resting-state in pre-reading children at risk for dyslexia. Three matched groups of pre-readers at risk for dyslexia were tested before and after they played with AVG, non-AVG (for 20 hrs) or no-treatment (Exp. 1). We found that only playing AVG improved children's visuo-spatial attention processing. Phonemes discrimination was also increased only after AVG training. We confirmed this effect of AVG training on phonological processing in a replication study with another independent sample (Exp. 2). Two new samples of pre-schoolers at risk for dyslexia were selected (Exp. 3): half of them were trained with AVG while the other half had no training. We measured visuo-spatial attention and auditory-phonological skills. Eyes-closed resting-state EEG was also recorded in both groups. Results showed that only the AVG training improved visuo-spatial attention as well as auditory-phonological skills. A reduction of the upper alpha band (10-14 Hz) oscillatory activity in posterior areas was found only after the AVG training, showing a possible neural basis of the effect of attentional improvement on auditory-phonological processing. Our results showed, for the first time, that attention improvements can directly translate into better language abilities, providing a new, fast and fun prevention training for dyslexia.