OPA1 is a ubiquitously expressed, nuclear dynamin-related GTPase, targeted to the inner mitochondrial membrane, which plays a role in mitochondrial fusion. Mutations in the OPA1 gene on chromosome 3q28-qter are associated with autosomal dominant optic atrophy (ADOA), the most common inherited optic neuropathy, in which retinal ganglion cells (RGCs) are lost and visual acuity is impaired from an early age. We have generated a novel ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in opa1 in order to explore the pathophysiology of ADOA. The heterozygous mutation, B6; C3-Opa1(Q285STOP), located in exon 8 immediately before the central dynamin-GTPase, leads to approximately 50% reduction in opa1 protein in retina and all tissues on western analysis. The homozygous mutation is embryonic lethal by 13.5 days post coitum, demonstrating the importance of Opa1 during early development. Fibroblasts taken from adult heterozygous mutant mice show an apparent alteration in morphology, with an increase in mitochondrial fission and fragmentation. Heterozygous mutants show a slow onset of degeneration in the optic nerve electron microscopy. Furthermore, they demonstrate a functional reduction in visual function on testing with the optokinetic drum and the circadian running wheel. These findings indicate that the opa1 GTPase contains crucial information required for the survival of RGCs and that Opa1 is essential for early embryonic survival. The Opa1 +/- mice described here provide a means to directly investigate the cellular pathophysiology of OPA1 ADOA.
Human alveolar type II cells were isolated from lung tissue and cultured for several days. The morphology of cells was investigated at different time points postseeding and the synthesis of alveolar cell-type specific proteins was analyzed using different methods. The rationale of the study was to characterize a primary cell culture of human alveolar cells for the development of an in vitro model studying pulmonary drug delivery. In vitro test systems based on human cells are attracting increasing interest as important alternatives to animal-derived models because possible interspecies differences in alveolar cell biology and transport mechanisms cannot be excluded. In our study, both morphological characterization and marker protein synthesis of human alveolar cells in culture indicate the differentiation of isolated alveolar type II cells into epithelial monolayers consisting of alveolar type I-like and alveolar type II-like cells, which corresponds to the composition of the alveolar epithelium of the donor tissue. By using flow cytometry, immunofluorescence, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR), we observed a shift in the synthesis of important marker proteins. Early cultures were characterized by low caveolin-1 and high Sp-C levels. In comparison, the protein biosynthesis of alveolar cells switched with time of culture to high caveolin-1 and low Sp-C levels. Based on the similarity between human alveolar epithelium and the development of our primary alveolar cell culture, we suggest that the culture may serve as a suitable model to study epithelial transport or cell biological processes in human alveolar cells.
Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.
The multidrug resistant (MDR) transporter P-glycoprotein (Pgp) is constitutively expressed in normal tissues, where its spatial distribution defines it as an important element reducing the systemic exposure and tissue access of potentially harmful xenobiotics. We sought to determine whether P-gp is functionally expressed within alveolar epithelium of lung, in particular within the predominant cell type of this barrier, the alveolar epithelial (AE) type I cell. By immunohistochemistry, MDR-1/ mdr-1 P-gp was localized to luminal membranes of AE type I epithelium within normal human and rat lung tissue. Using a primary rat cell culture model affording study of AE type II to AE type I differentiation, we observed increased expression (reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunoflow cytometry techniques) of mdr-1a and mdr-1b P-gp in the cultures as they adopted an AE type I phenotype; freshly isolated AE type II cells were negative for mdr-1/P-gp. The functionality of P-gp within the AE cultures was demonstrated by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE type I monolayers (basal-to-apical permeability was 3-fold that of apical-to-basal permeability), which was found to be comparable with the P-gp transport barrier presented by Caco-2 cell monolayers. The implications of localizing P-gp within alveolar epithelium is of significance to studies of fundamental respiratory cell biology as well as to further clarifying the nature of the barrier to xenobiotic transfer from alveolar airspace to pulmonary interstitium and capillary blood.P-Glycoprotein (P-gp) is a member of the ATP-binding cassette superfamily of membrane transport proteins that mediates the vectorial movement across cell membranes of a wide range of physicochemically diverse solutes (Stouch and Gudmundsson, 2002). In humans, two P-gp-related genes have been cloned and subsequently termed MDR1 and MDR3 (for review, see Ambudkar et al., 1999). The MDR1/P-gp gene product is recognized in particular to actively efflux from a cell a diverse range of cytotoxic drugs, a characteristic that is an important facet in the multidrug resistant (MDR) cell phenotype. Current evidence suggests that the MDR3/P-gp gene product does not contribute to an MDR phenotype.In rodents, three P-gp-related genes have been identified and designated mdr-1a, mdr-1b, and mdr-
A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro.
Polypropylenimine (PPI) dendrimers appear attractive non-viral vectors for the delivery of genes, antisense oligonucleotides, and small interfering RNA (siRNA). However, the effects of these synthetic gene delivery vectors on global gene expression are poorly understood. Here we have examined the toxicogenomics of generation 2 (DAB-8) and generation 3 (DAB-16) PPI dendrimers in two human cell lines. At concentrations and treatment protocols routinely used for gene and oligonucleotide transfection, PPI dendrimers alone elicited marked changes in endogenous gene expression in A431 epithelial cells. The extent of PPI-induced gene changes appeared to be dependent on the dendrimer generation as the number of genes affected was greater with G3 compared to G2 PPI dendrimers in A431 cells. The signature of DAB16-induced gene changes in A549 cells was different to those elicited in A431 cells implying a strong dependence on cell type. The DAB-16 polymer complexed with DNA (dendriplexes) also elicited marked gene expression changes in A549 cells but with a signature that was different from the polymer alone implying that dendriplexes are "recognised" by cells as chemical entities that are distinct from the polymer alone. Alterations in expression of a variety of gene ontologies were observed including those involved in defence responses, cell proliferation and apoptosis. Although there was a tendency for increased DNA damage in cells treated with DAB16 alone or its DNA dendriplexes as detected by the COMET assay, these differences were not statistically significant. These data show for the first time that PPI-dendrimers, separate from their capability as transfection reagents, can intrinsically alter the expression of many endogenous genes that could potentially lead to them exerting multiple biological effects in cells. The impact and consequences of polymer-induced gene changes should guide their rational use as delivery systems for gene-based therapeutics.
RNA interference (RNAi) represents a promising new gene silencing technology for functional genomics and a potential therapeutic strategy for a variety of genetic diseases. RNAi involves the targeted post-transcriptional degradation of messenger RNA thereby inhibiting the synthesis of the desired protein. This effectively leads to silencing of gene expression. The effectors of this process are short interfering RNA (siRNA) duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytosol. However, due to the large molecular weight and negative charge of siRNA duplexes the effective cellular uptake and intracellular delivery appear to represent a major challenge for the widespread use of RNAi in vivo. This review summarises some of the main delivery strategies that have been attempted for the transfection of siRNA to cells in vitro and in vivo.
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