BackgroundMutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays.ResultsWe have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression.ConclusionOur data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

Astrocyte-secreted peptidergic and non-peptidergic transmitters regulate development, physiology and pathology of neuronal circuits (Araque et al. 2001;Volterra and Meldolesi 2005). For instance, astroglial-derived thrombospondins and ApoE-containing lipoproteins control synaptogenesis, while astrocytic glutamate, ATP and D-serine modulate synaptic transmission and network activity (Mauch et al. 2001;Fellin et al. 2004;Christopherson et al. 2005;Pascual et al. 2005;Panatier et al. 2006;Perea and Araque 2007). Moreover, proinflammatory cytokines, growth factors and amino acids released from glia have been associated with neurological disorders and neurodegenerative diseases ( Address correspondence and reprint requests to Dr Fernando Aguado, Department of Cell Biology, University of Barcelona, Av. Diagonal 645, Barcelona E-08028, Spain. E-mail: faguado@ub.eduAbbreviations used: BAPTA, 1,2-bis(2-amino-phenoxy)ethane-N, N, N¢, N¢-tetraacetic acid; GFAP, glial fibrillary acidic protein; GFP, green flourescent protein; IFN, interferon; IL, interleukin; PBS, phosphatebuffered saline; SCG-2, secretogranin-2; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SNAP, synaptosomal-associated protein; SNAREs, N-ethylmaleimide-sensitive factor attachment protein receptors; TNF, tumor necrosis factor; TPA, phorbol-12-myristate-13-acetate; VAMP, vesicle-associated membrane protein. AbstractVesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca 2+ -dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca 2+ ] i . As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca 2+ -regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca 2+ -dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.

Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressorRB1. VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.

Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.