Laura Di Francesco

Nuclear accumulation of mRNAs underlies G4C2 repeat-induced translational repression in a cellular model of C9orf72 ALS

A common feature of non‐coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA‐binding proteins, thus affecting various steps of post‐transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur‐alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra‐cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.

Conus ventricosus venom peptides profiling by HPLC-MS: a new insight in the intraspecific variation.

Conus is a genus of predatory marine gastropods that poison the prey with a complex mixture of compounds active on muscle and nerve cells. An individual cone snails venom contains a mixture of pharmacological agents, mostly short, structurally constrained peptides. This study is focused on the composition of the venom employed by Conus ventricosus Gmelin, 1791, a worm-hunting cone snail living in the Mediterranean Sea. For this purpose, LC coupled to MS techniques has been successfully used to establish qualitative and quantitative differences in conopeptides from minute amounts of venom ducts. We were able to prove variability in the venom conopeptide complement, possibly related to different trophic habits of the species in the Mediterranean Sea. Moreover, the information-rich MS techniques enabled us to identify two novel C. ventricosus peptides, here named Conotoxin-Vn and -Conotoxin-Vn. On the basis of the structural data collected so far, we suggest that Conotoxin-Vn is a conopeptide belonging to the -family that recognizes calcium channels through a specific pharmacophore. Similarly, molecular modeling data suggest that -Conotoxin-Vn should represent a competitive antagonist of neuronal nicotinic acetylcholine receptors (nAChRs).

Importin-β negatively regulates multiple aspects of mitosis including RANGAP1 recruitment to kinetochores

Importin-β binds RANBP2 and RANGAP1 and antagonizes CRM1 activity during RANGAP1 recruitment to kinetochores.

14‐3‐3ε marks the amyloid‐stimulated microglia long‐term activation

Microglia‐mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimers disease. Microglial cells activation follows the deposition of amyloid β fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimers disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid β peptides in triggering microglial activation. The response was monitored at the proteome level through a two‐dimensional gel electrophoresis‐based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14‐3‐3ε protein, found significantly upregulated in Aβ‐activated cells. 14‐3‐3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.

The pathological prion protein forms ionic conductance in lipid bilayer

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.

Acetylation and phosphorylation of STAT3 are involved in the responsiveness of microglia to beta amyloid

Microglia are macrophages within the central nervous system playing a central role in neurodegenerative disorders. Although the initial engagement of microglia seems to be neuroprotective, many lines of evidence indicate that its persistent activation contributes to dismantle neuronal activity and to induce neuronal loss. The molecular pathways that lead from amyloid interaction with membrane receptors to the microglial activation have been extensively investigated, although a definitive picture is not yet at hand. In this work, primary and immortalized microglial cells were treated with a synthetic form of Aβ peptides, and relative abundance of acetylated and phosphorylated STAT3 were assayed. Results highlight, for the first time, three distinctive sequential events: i) an earlier event marked by the increase in the level of STAT3 acetylated species, followed by ii) a later increase in the level of STAT3 phosphorylated form, and finally iii) an involvement of phosphorylated STAT3 in the increase in expression of the 14-3-3 epsilon, a protein frequently associated with neurodegenerative diseases and known to be a marker of Aβ-activated microglia. These data outline a complex, time-dependent modification of STAT3 signalling triggered by amyloid in the microglial compartments, that once confirmed by in vivo experiments will broaden the knowledge of the molecular basis of amyloid neurotoxicity.

Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively.

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt-Jakob disease patients.

Creutzfeldt-Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrP(CJD)) of the host encoded cellular prion protein (PrP(C)). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrP(CJD) allotypes in brains from affected subjects. We found that the mutant PrP(CJD) allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade. Different mechanisms can be hypothesized to explain the pathogenic role of mutant residues: V210I and R208H substitutions can increase the concentration of PrP(C) and the probability to form insoluble aggregates, or they may facilitate the formation of pathological intermediates, or, alternatively, they may increase the affinity for ligands that are involved in the initial phases of PrP(CJD) formation and aggregation. Whatever the mechanism, the enrichment found for the mutated PrP(CJD) species indicates that these altered structures are more prone, with respect to the non-mutated ones, to be captured in the polymerization process either at the onset or during the development of the disease.

Plasma membrane protein profiling in beta‐amyloid‐treated microglia cell line

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi‐stage aqueous two‐phase partition system. We were able to identify by 1D‐LC‐MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24‐h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity‐regulating kinase 3 (MARK3), Interferon‐induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin‐1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.