FMRP expression in primary breast tumor cells correlates with recurrence and specific site of metastasis.

Breast cancer is the most common cancer among women worldwide. Molecular and clinical evidence indicated that Fragile X Messenger Ribonucleoprotein 1 (FMRP) plays a role in different types of cancer, including breast cancer. FMRP is an RNA binding protein that regulates the metabolism of a large group of mRNAs coding for proteins involved in both neural processes and in epithelial-mesenchymal transition, a pivotal mechanism that in cancer is associated to tumor progression, aggressiveness and chemoresistance. Here, we carried out a retrospective case-control study of 127 patients, to study the expression of FMRP and its correlation with metastasis formation in breast cancer. Consistent with previous findings, we found that FMRP levels are high in tumor tissue. Two categories have been analyzed, tumor with no metastases (referred as control tumors, 84 patients) and tumor with distant metastatic repetition, (referred as cases, 43 patients), with a follow-up of 7 years (mean). We found that FMRP levels were lower in both the nuclei and the cytoplasm in the cases compared to control tumors. Next, within the category cases (tumor with metastases) we evaluated FMRP expression in the specific sites of metastasis revealing a nuclear staining of FMRP. In addition, FMRP expression in both the nuclear and cytoplasmic compartment was significantly lower in patients who developed brain and bone metastases and higher in hepatic and pulmonary sites. While further studies are required to explore the underlying molecular mechanisms of FMRP expression and direct or inverse correlation with the secondary metastatic site, our findings suggest that FMRP levels might be considered a prognostic factor for site-specific metastasis.

A white paper on a neurodevelopmental framework for drug discovery in autism and other neurodevelopmental disorders.

In the last decade there has been a revolution in terms of genetic findings in neurodevelopmental disorders (NDDs), with many discoveries critical for understanding their aetiology and pathophysiology. Clinical trials in single-gene disorders such as fragile X syndrome highlight the challenges of investigating new drug targets in NDDs. Incorporating a developmental perspective into the process of drug development for NDDs could help to overcome some of the current difficulties in identifying and testing new treatments. This paper provides a summary of the proceedings of the 'New Frontiers Meeting' on neurodevelopmental disorders organised by the European College of Neuropsychopharmacology in conjunction with the Innovative Medicines Initiative-sponsored AIMS-2-TRIALS consortium. It brought together experts in developmental genetics, autism, NDDs, and clinical trials from academia and industry, regulators, patient and family associations, and other stakeholders. The meeting sought to provide a platform for focused communication on scientific insights, challenges, and methodologies that might be applicable to the development of CNS treatments from a neurodevelopmental perspective. Multidisciplinary translational consortia to develop basic and clinical research in parallel could be pivotal to advance knowledge in the field. Although implementation of clinical trials for NDDs in paediatric populations is widely acknowledged as essential, safety concerns should guide each aspect of their design. Industry and academia should join forces to improve knowledge of the biology of brain development, identify the optimal timing of interventions, and translate these findings into new drugs, allowing for the needs of users and families, with support from regulatory agencies.

Altered neuronal network and rescue in a human MECP2 duplication model.

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders.

Autism Spectrum Disorders: Translating human deficits into mouse behavior.

Autism Spectrum Disorders are a heterogeneous group of neurodevelopmental disorders, with rising incidence but little effective therapeutic intervention available. Currently two main clinical features are described to diagnose ASDs: impaired social interaction and communication, and repetitive behaviors. Much work has focused on understanding underlying causes of ASD by generating animal models of the disease, in the hope of discovering signaling pathways and cellular targets for drug intervention. Here we review how ASD behavioral phenotypes can be modeled in the mouse, the most common animal model currently in use in this field, and discuss examples of genetic mouse models of ASD with behavioral features that recapitulate various symptoms of ASD.

Acetylation of RTN-1C regulates the induction of ER stress by the inhibition of HDAC activity in neuroectodermal tumors.

Reticulons are a family of highly conserved proteins, localized in the endoplasmic reticulum (ER) and involved in different cellular functions, such as intracellular membrane trafficking, apoptosis and nuclear envelope formation. The reticulon protein family consists of four members, but their specific functions are presently poorly understood. RTN-1C overexpression triggers apoptosis, regulating ER stress versus DNA damage-induced cell death in a mutually exclusive way. The different RTN isoforms share a C-terminal reticulon homology domain containing two hydrophobic segments and a 66-amino acid hydrophilic loop. In the C-terminal region of RTN-1C, a unique consensus sequence (GAKRH) has recently been identified, showing 100% identity with the DNA-binding domain of histone H4. In this study, we show that this sequence is essential for RTN-1C-mediated apoptosis. It is noteworthy that the lysine 204 present in this region is post-translationally modified by acetylation and that this event is associated with a significant decrease in histone deacetylase activity and contributes to RTN-1C binding to DNA. These data demonstrate a molecular mechanism by which RTN-1C controls apoptosis and indicate this protein to be a novel potential target for cancer therapy.

Cortical and subcortical distribution of ionotropic purinergic receptor subunit type 1 (P2X(1)R) immunoreactive neurons in the rat forebrain.

Ionotropic purinergic receptors (P2XR) are ATP-gated cationic channels composed of seven known subunits (P2X(1-7)R) and involved in different functions in neural tissue. Although their presence has been demonstrated in the brain, few studies have investigated their expression pattern. In particular, ionotropic purinergic receptor subunit type 1 (P2X(1)R) has been observed in the cerebellum and in brainstem nuclei. The present study investigates the P2X(1)R expression pattern in the rat forebrain using immunohistochemistry. The specificity of the immunolabeling has been verified by Western blotting and in situ hybridization methods. P2X(1)R immunoreactivity was specifically localized in neurons, dendrites and axons throughout the forebrain. Characteristic differences in the distribution of P2X(1)R were observed in different cortical areas. In prefrontal, cingulate and perirhinal cortices, very intense labeling was present in neuronal bodies. In frontal, parietal, temporal and occipital cortices, immunostaining was lighter and mainly found in dendrites and axons. The hippocampal formation was intensely labeled. Labeling was present almost exclusively in dendrites and axons and never in neuronal bodies. The diencephalon was devoid of P2X(1)R positive neurons or fibers except for the medial habenular nucleus, which showed very intense P2X(1)R immunostaining. Furthermore, two subcortical regions, namely, the nucleus centralis of the amygdala and the bed nucleus of the stria terminalis, showed intense P2X(1)R neuronal labeling. Present data indicate that P2X(1)R are prevalent in forebrain areas involved in the integration of cognitive, limbic and autonomic functions.

Role of atrial natriuretic peptide in the suppression of lysophosphatydic acid-induced rat aortic smooth muscle (RASM) cell growth.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. In the present study we investigated the possible role of atrial natriuretic peptide (ANP), a hormone affecting cardiovascular homeostasis and inducing antimitogenic effects in different cell types, on LPA-induced cell growth and reactive oxygen species (ROS) production in rat aortic smooth muscle (RASM) cells. Both LPA effects on cell growth and levels of ROS were totally abrogated by physiological concentrations of ANP, without modifying the overexpression of LPA-receptors. These effects were also affected by cell pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Moreover, the LPA-induced activation of Akt, a downstream target of PI3K, was completely inhibited by physiological concentrations of ANP, which were also able to inhibit p42/p44 phosphorylation. Taken together, our data suggest that PI3K may represent an important step in the LPA signal transduction pathway responsible for ROS generation and DNA synthesis in RASM cells. At same time, the enzyme could also represent an essential target for the antiproliferative effects of ANP.

Familial adenomatous polyposis. Surgical treatment: when and how.

Familial adenomatous polyposis is an autosomal dominated inherited disease, caused by the mutation of the tumour suppressor gene adenomatous polyposis coli on chromosome 5. Despite being a rare disorder, accounting for some 1% of colorectal cancers, it represents an interesting model of hereditary disease, because of its intrinsic characteristics, conventionally defined by the presence of more than 100 colorectal polyps, as well as extra-colon manifestations, the attenuated form of the disease, genetic aspects, the inevitable progression to colorectal cancer and hence the correct therapy to treat or prevent the fatal evolution of the disease. Surgical treatment is based above all on two techniques: ileorectal anastomosis, which requires careful surveillance of rectal remnant, and ileal pouch-anal anastomosis, which totally eradicates the disease. The suitability of using these two techniques is discussed in view of new genetic and clinical findings, acquired from personal experience and from the literature.

A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P.

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP--CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

Fibrillin-1 (FBN1) gene frameshift mutations in Marfan patients: genotype-phenotype correlation.

Marfan syndrome (MFS) is a multisystemic disease associated with mutations in the fibrillin-1 gene. Most of the reported mutations are missense substitutions mainly affecting the epidermal growth factor (EGF)-like protein domain structure and the calcium-binding (cb) site. The aim of our study was to investigate the correlation between fibrillin-1 frameshift mutations and the clinical phenotype in patients affected by MFS. In 48 out of 66 Marfan patients a pathogenetic mutation was found. We detected novel mutations causing premature termination codon in exons 19, 37, 40 and 41 of four Italian patients. The first mutation in exon 19 (cbEGF #8 domain) results in a clinical phenotype involving mainly the skeletal and cardiovascular systems. Interestingly, we noticed that, while mutations in exons 37 and 41 (eight cysteine domains #4 and #5) are milder, the mutation in exon 40 (cbEGF #24 domain) is more severe and causes major cardiovascular involvement with thoracic and abdominal aortic aneurysms. It is noteworthy that the degree of the severity in the phenotype of one of our patients and another from the literature carrying a mutation in exon 41 could be explained with alterations in mRNA expression.

Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues.

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.

Repair of giant hernias using more prosthesis.

Giant incisional hernias with total loss of substance are an ominous pathological condition characterized by massive depletion of muscular and fascial tissue, by complete loss of the anatomical and physiological function of the abdominal wall and by severe respiratory and visceral involvement. Over a 10-year period we operated 270 patients with voluminous incisional hernias, 12 of which had a total loss of substance. There was no intraoperative mortality. One patient died of myocardial infarction on the fifth and one died of intestinal occlusion and peritonitis the 11th postoperative day. Early postoperative complications occurred in only one patient who had skin necrosis with an infection at the polypropylene mesh. This was successfully treated with systemic antibiotic therapy and topical medication of the wound. There was also one minor recurrence over the pubis 1 year after the operation that required a new operation to replace the mesh. No respiratory complications occurred and all patients were normally active. The good results reported in our series encourage us to continue in this direction even though these patients are at high risk.

Chemical stimulation of synaptosomes modulates alpha -Ca2+/calmodulin-dependent protein kinase II mRNA association to polysomes.

The presence of specific mRNAs in dendrites and at synapses is well established, but a direct and reliable demonstration that they are associated with polysomes is still missing. To address this point we analyzed the polysomal association of the mRNAs for the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (alpha-CaMKII), for type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) and for the activity-regulated cytoskeleton-associated protein (Arc) in a synaptosomal preparation devoid of contaminating material from neuronal and glial perikarya. We show that a fraction of alpha-CaMKII, InsP3R1, and Arc mRNAs present in synaptosomes is indeed associated with polysomes. Moreover, we show that polysomal association of alpha-CaMKII mRNA, but not InsP3R1 and Arc mRNAs, increases with depolarization of the synaptosomal membrane. Finally, we show that the synthesis of alpha-CaMKII protein increases with stimulation. Dendritic mRNA recruitment onto polysomes in response to synaptic stimulation might represent one of the mechanisms underlying the processes of learning and memory.

Simple and complicated acute appendicitis. Indications for a correct diagnosis.

BACKGROUND: In this retrospective investigation the symptoms, signs, and laboratory findings collected in 2 groups of patients with simple and complicated acute appendicitis, respectively, have been observed in order to give some indication for a correct diagnosis and surgical treatment. METHODS: A total of 103 consecutive patients affected by simple and complicated acute appendicitis submitted to surgical operation have been studied. RESULTS: Data collected show statistically significant differences between clinical presentation of simple and complicated acute appendicitis. CONCLUSIONS: The conclusion is draws in that anamnesis and clinical examination of the patients affected by acute appendicitis are the best indications for an exact diagnosis and to select patients who need an immediate operation.

Expression of the gene for mitoribosomal protein S12 is controlled in human cells at the levels of transcription, RNA splicing, and translation.

The human gene RPMS12 encodes a protein similar to bacterial ribosomal protein S12 and is proposed to represent the human mitochondrial orthologue. RPMS12 reporter gene expression in cultured human cells supports the idea that the gene product is mitochondrial and is localized to the inner membrane. Human cells contain at least four structurally distinct RPMS12 mRNAs that differ in their 5'-untranslated region (5'-UTR) as a result of alternate splicing and of 5' end heterogeneity. All of them encode the same polypeptide. The full 5'-UTR contains two types of sequence element implicated elsewhere in translational regulation as follows: a short upstream open reading frame and an oligopyrimidine tract similar to that found at the 5' end of mRNAs encoding other growth-regulated proteins, including those of cytosolic ribosomes. The fully spliced (short) mRNA is the predominant form in all cell types studied and is translationally down-regulated in cultured cells in response to serum starvation, even though it lacks both of the putative translational regulatory elements. By contrast, other splice variants containing one or both of these elements are not translationally regulated by growth status but are translated poorly in both growing and non-growing cells. Reporter analysis identified a 26-nucleotide tract of the 5'-UTR of the short mRNA that is essential for translational down-regulation in growth-inhibited cells. Such experiments also confirmed that the 5'-UTR of the longer mRNA variants contains negative regulatory elements for translation. Tissue representation of RPMS12 mRNA is highly variable, following a typical mitochondrial pattern, but the relative levels of the different splice variants are similar in different tissues. These findings indicate a complex, multilevel regulation of RPMS12 gene expression in response to signals mediating growth, tissue specialization, and probably metabolic needs.

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.

Novel RNA-binding motif: the KH module.

The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH-containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed.