Interleukin-6 neutralization ameliorates symptoms in prematurely aged mice.

Hutchinson-Gilford progeria syndrome (HGPS) causes premature aging in children, with adipose tissue, skin and bone deterioration, and cardiovascular impairment. In HGPS cells and mouse models, high levels of interleukin-6, an inflammatory cytokine linked to aging processes, have been detected. Here, we show that inhibition of interleukin-6 activity by tocilizumab, a neutralizing antibody raised against interleukin-6 receptors, counteracts progeroid features in both HGPS fibroblasts and LmnaG609G/G609G progeroid mice. Tocilizumab treatment limits the accumulation of progerin, the toxic protein produced in HGPS cells, rescues nuclear envelope and chromatin abnormalities, and attenuates the hyperactivated DNA damage response. In vivo administration of tocilizumab reduces aortic lesions and adipose tissue dystrophy, delays the onset of lipodystrophy and kyphosis, avoids motor impairment, and preserves a good quality of life in progeroid mice. This work identifies tocilizumab as a valuable tool in HGPS therapy and, speculatively, in the treatment of a variety of aging-related disorders.

The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations.

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Targeting the diuretic hormone receptor to control the cotton leafworm, Spodoptera littoralis.

The cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), is one of the most devastating pests of crops worldwide. Several types of treatments have been used against this pest, but many of them failed because of the rapid development of genetic resistance in the different insect populations. G protein coupled receptors have vital functions in most organisms, including insects; thus, they are appealing targets for species-specific pest control strategies. Among the insect G protein coupled receptors, the diuretic hormone receptors have several key roles in development and metabolism, but their importance in vivo and their potential role as targets of novel pest control strategies are largely unexplored. With the goal of using DHR genes as targets to control S. littoralis, we cloned a corticotropin-releasing factor-like binding receptor in this species and expressed the corresponding dsRNA in tobacco plants to knock down the receptor activity in vivo through RNA interference. We also expressed the receptor in mammalian cells to study its signaling pathways. The results indicate that this diuretic hormone receptor gene has vital roles in S. littoralis and represents an excellent molecular target to protect agriculturally-important plants from this pest.

Egfr signaling modulates VM32E gene expression during Drosophila oogenesis.

Drosophila vitelline membrane gene VM32E is expressed in the follicle cells of the stage 10 egg chamber and shows a peculiar temporal and spatial expression pattern compared to the other members of the same gene family. Previous work has led us to demonstrate that Decapentaplegic (Dpp) signaling represses the expression of the VM32E gene in the centripetal follicle cells. In this paper, we describe another level of complexity of the VM32E gene expression regulation. Through clonal analyses, we show that the expression of the VM32E gene in the main body follicle cells is modulated by the epidermal growth factor receptor (Egfr) activity. In follicle cell clones expressing a constitutively active form of the Egfr, the VM32E gene is downregulated, while the loss of the Egfr activity upregulates VM32E expression. In addition, we show that the ectopic expression of the Egfr-induced ETS transcription factor PointedP2 (PntP2) affects the expression of the VM32E gene. From these results and our previously published data, it appears that the proper patterning of follicle cells, defined by Dpp and Egfr signaling pathways, controls the VM32E gene expression pattern. This may suggest that a fine tuning of the expression of specific eggshell structural genes could be part of the complex process that leads to a proper eggshell assembly.

Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1.

BACKGROUND: Anosmin-1, the protein implicated in the X-linked Kallmann's syndrome, plays a role in axon outgrowth and branching but also in epithelial morphogenesis. The molecular mechanism of its action is, however, widely unknown. Anosmin-1 is an extracellular protein which contains a cysteine-rich region, a whey acidic protein (WAP) domain homologous to some serine protease inhibitors, and four fibronectin-like type III (FnIII) repeats. Drosophila melanogaster Kal-1 (DmKal-1) has the same protein structure with minor differences, the most important of which is the presence of only two FnIII repeats and a C-terminal region showing a low similarity with the third and the fourth human FnIII repeats. We present a structure-function analysis of the different DmKal-1 domains, including a predicted heparan-sulfate binding site. RESULTS: This study was performed overexpressing wild type DmKal-1 and a series of deletion and point mutation proteins in two different tissues: the cephalopharyngeal skeleton of the embryo and the wing disc. The overexpression of DmKal-1 in the cephalopharyngeal skeleton induced dosage-sensitive structural defects, and we used these phenotypes to perform a structure-function dissection of the protein domains. The reproduction of two deletions found in Kallmann's Syndrome patients determined a complete loss of function, whereas point mutations induced only minor alterations in the activity of the protein. Overexpression of the mutant proteins in the wing disc reveals that the functional relevance of the different DmKal-1 domains is dependent on the extracellular context. CONCLUSION: We suggest that the role played by the various protein domains differs in different extracellular contexts. This might explain why the same mutation analyzed in different tissues or in different cell culture lines often gives opposite phenotypes. These analyses also suggest that the FnIII repeats have a main and specific role, while the WAP domain might have only a modulator role, strictly connected to that of the fibronectins.

Embryonic expression pattern of the Drosophila Kallmann syndrome gene kal-1.

The role of kal-1, the gene responsible for the X chromosome-linked form of Kallmann syndrome, is not well definite. In Drosophila, the kal-1 gene encodes a putative protein with the characteristic kal-1 topology but with only two Fibronectin-like type III (FnIII) domains. We studied the embryonic expression pattern of kal-1 using whole mount in situ hybridization. This gene is expressed in the second half of embryogenesis showing a complex and dynamic pattern. kal-1 is expressed during important morphogenetic processes such as germ band retraction, dorsal closure and head involution. We found expression in cells associated with different sensory organs, such as the antennal organ, which has an olfactory function, the chordotonal organ, the Keilin's organ and the dorsal pharyngeal organ. Expression of kal-1 in the head also regards some ectodermal cells of the gnathal lobes. By studying the expression in Dfd and cnc homeotic mutants, we found that these ectodermal cells derive from the anterior and posterior mandibular segment, whose determination depends on cnc, and that the expression in the posterior mandibular segment requires Dfd activity. kal-1 is also expressed in the posterior part of the male gonads in a specific subset of the somatic cells called male-specific somatic gonadal precursors (msSGPs). This is the first time that the expression of a kal-1 ortholog has been demonstrated to be sex specific making the kal-1 transcript a useful tool for the study of sex determination in the gonad.