SMN Deficiency Destabilizes ABCA1 Expression in Human Fibroblasts: Novel Insights in Pathophysiology of Spinal Muscular Atrophy.

The deficiency of survival motor neuron protein (SMN) causes spinal muscular atrophy (SMA), a rare neuromuscular disease that affects different organs. SMN is a key player in RNA metabolism regulation. An intriguing aspect of SMN function is its relationship with plasma membrane-associated proteins. Here, we provide a first demonstration that SMN affects the ATP-binding cassette transporter A1, (ABCA1), a membrane protein critically involved in cholesterol homeostasis. In human fibroblasts, we showed that SMN associates to ABCA1 mRNA, and impacts its subcellular distribution. Consistent with the central role of ABCA1 in the efflux of free cholesterol from cells, we observed a cholesterol accumulation in SMN-depleted human fibroblasts. These results were also confirmed in SMA type I patient-derived fibroblasts. These findings not only validate the intimate connection between SMN and plasma membrane-associated proteins, but also highlight a contribution of dysregulated cholesterol efflux in SMA pathophysiology.

Identification of protein/mRNA network involving the PSORS1 locus gene CCHCR1 and the PSORS4 locus gene HAX1.

CCHCR1 (Coiled-Coil alpha-Helical Rod 1), maps to chromosomal region 6p21.3, within the major psoriasis susceptibility locus PSORS1. CCHCR1 itself is a plausible psoriasis candidate gene, however its role in psoriasis pathogenesis remains unclear. We previously demonstrated that CCHCR1 protein acts as a cytoplasmic docking site for RNA polymerase II core subunit 3 (RPB3) in cycling cells, suggesting a role for CCHCR1 in vesicular trafficking between cellular compartments. Here, we report a novel interaction between CCHCR1 and the RNA binding protein HAX1. HAX1 maps to chromosomal region 1q21.3 within the PSORS4 locus and is over-expressed in psoriasis. Both CCHCR1 and HAX1 share subcellular co-localization with mitochondria, nuclei and cytoplasmic vesicles as P-bodies. By a series of ribonucleoprotein immunoprecipitation (RIP) assays, we isolated a pool of mRNAs complexed with HAX1 and/or CCHCR1 proteins. Among the mRNAs complexed with both CCHCR1 and HAX1 proteins, there are Vimentin mRNA, previously described to be bound by HAX1, and CAMP/LL37 mRNA, whose gene product is over-expressed in psoriasis.

Utrophin up-regulation by artificial transcription factors induces muscle rescue and impacts the neuromuscular junction in mdx mice.

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy (DMD). In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZF-ATFs) that target the utrophin 'A' promoter. We have previously shown that the ZF-ATF "Jazz", either by transgenic manipulation or by systemic adeno-associated viral delivery, induces significant rescue of muscle function in dystrophic "mdx" mice. We present the full characterization of an upgraded version of Jazz gene named "JZif1" designed to minimize any possible host immune response. JZif1 was engineered on the Zif268 gene-backbone using selective amino acid substitutions to address JZif1 to the utrophin 'A' promoter. Here, we show that JZif1 induces remarkable amelioration of the pathological phenotype in mdx mice. To investigate the molecular mechanisms underlying Jazz and JZif1 induced muscle functional rescue, we focused on utrophin related pathways. Coherently with utrophin subcellular localization and role in neuromuscular junction (NMJ) plasticity, we found that our ZF-ATFs positively impact the NMJ. We report on ZF-ATF effects on post-synaptic membranes in myogenic cell line, as well as in wild type and mdx mice. These results candidate our ZF-ATFs as novel therapeutic molecules for DMD treatment.

eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts.

BACKGROUND: We have previously shown that the eukaryotic elongation factor subunit 1B gamma (eEF1Bγ) interacts with the RNA polymerase II (pol II) alpha-like subunit "C" (POLR2C), alone or complexed, in the pol II enzyme. Moreover, we demonstrated that eEF1Bγ binds the promoter region and the 3' UTR mRNA of the vimentin gene. These events contribute to localize the vimentin transcript and consequentially its translation, promoting a proper mitochondrial network. METHODS: With the intent of identifying additional transcripts that complex with the eEF1Bγ protein, we performed a series of ribonucleoprotein immunoprecipitation (RIP) assays using a mitochondria-enriched heavy membrane (HM) fraction. RESULTS: Among the eEF1Bγ complexed transcripts, we found the mRNA encoding the Che-1/AATF multifunctional protein. As reported by other research groups, we found the tumor suppressor p53 transcript complexed with the eEF1Bγ protein. Here, we show for the first time that eEF1Bγ binds not only Che-1 and p53 transcripts but also their promoters. Remarkably, we demonstrate that both the Che-1 transcript and its translated product localize also to the mitochondria and that eEF1Bγ depletion strongly perturbs the mitochondrial network and the correct localization of Che-1. In a doxorubicin (Dox)-induced DNA damage assay we show that eEF1Bγ depletion significantly decreases p53 protein accumulation and slightly impacts on Che-1 accumulation. Importantly, Che-1 and p53 proteins are components of the DNA damage response machinery that maintains genome integrity and prevents tumorigenesis. CONCLUSIONS: Our data support the notion that eEF1Bγ, besides its canonical role in translation, is an RNA-binding protein and a key player in cellular stress responses. We suggest for eEF1Bγ a role as primordial transcription/translation factor that links fundamental steps from transcription control to local translation.

SMN affects membrane remodelling and anchoring of the protein synthesis machinery.

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis.

Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular Dystrophy.

Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X-linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase five (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray-based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to (1) increased lipid metabolism; (2) a switch towards slow oxidative fibers driven by the up-regulation of PGC-1α; (3) an increased protein synthesis efficiency; (4) a better actin network organization at Z-disk.

Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD.

UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible. RESULTS: We have previously shown that the zinc finger-based artificial transcriptional factor "Jazz" corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named "UtroUp", engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus. CONCLUSIONS: This novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.

The eEF1γ subunit contacts RNA polymerase II and binds vimentin promoter region.

Here, we show that the eukaryotic translation elongation factor 1 gamma (eEF1γ) physically interacts with the RNA polymerase II (pol II) core subunit 3 (RPB3), both in isolation and in the context of the holo-enzyme. Importantly, eEF1γ has been recently shown to bind Vimentin mRNA. By chromatin immunoprecipitation experiments, we demonstrate, for the first time, that eEF1γ is also physically present on the genomic locus corresponding to the promoter region of human Vimentin gene. The eEF1γ depletion causes the Vimentin protein to be incorrectly compartmentalised and to severely compromise cellular shape and mitochondria localisation. We demonstrate that eEF1γ partially colocalises with the mitochondrial marker Tom20 and that eEF1γ depletion increases mitochondrial superoxide generation as well as the total levels of carbonylated proteins. Finally, we hypothesise that eEF1γ, in addition to its role in translation elongation complex, is involved in regulating Vimentin gene by contacting both pol II and the Vimentin promoter region and then shuttling/nursing the Vimentin mRNA from its gene locus to its appropriate cellular compartment for translation.

The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

Genotoxicity induced by fine urban air particulate matter in the macrophages cell line RAW 264.7.

Recent studies support a participation of fine airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 microm in the effects of air pollutants on health. Particulate matter was collected in an urban area of L'Aquila during the winter 2004. Fine particulate samples were analyzed by X-ray photoelectron spectroscopy (XPS) to determine the chemical inventory of the aerosol particle surfaces and to evaluate the weight of characteristic functional groups of the most frequent carbon-containing organic pollutant compounds (C-C/C-H, C-O/C-N, C=O, COOH). The most important contributor to the mass of fine particulate matter was carbon. The overall purpose of this work was to determine the in vitro toxicity and genotoxicity of fine PM in cultured macrophages (RAW 264.7 cells) since the biological target of inhaled PM are the pulmonary epithelium and resident macrophages. In parallel in vitro toxicity assays were used including cell viability and apoptosis. Genotoxicity was evaluated by the micronucleus (MN) assay. The viability of macrophages was assessed by the MTT method; apoptosis by an ELISA test for programmed cell death (PCD) was determined after RAW 264.7 cells treatment. Concentration of 1, 3 and 10 microg/cm2 of fine particles induced micronuclei in a dose-dependent manner. We also compared the effects of fine PM with those of fine carbon black particles (CB) in similar doses. Fine carbon black particles were consistently less genotoxic than the fine atmospheric particles, suggesting that the contaminants adsorbed on them (i.e. carbon-containing organic compounds in addition to metal oxides and metal salts) are involved in genotoxicity. Fine PM reduced cellular proliferation. Overall, the results presented here demonstrate the utility of in vitro tests in mouse cells for testing genotoxicity of urban air particulate matter.