Biomedical subjects

Arpad Palfi

Publications and source records attributed to Arpad Palfi.

8 recordsLinked to original sources

PubMed2006

RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.

Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae were dissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.

Animals↗

PubMed2005

Multiple calmodulin mRNAs are selectively transported to functionally different neuronal and glial compartments in the rat hippocampus. An electron microscopic in situ hybridization study.

The ultrastructural distribution of the calmodulin (CaM) mRNAs transcribed from the three CaM genes was studied in the CA1 region of the adult rat hippocampus by means of electron microscopic in situ hybridization. Digoxigenin-labeled CaM gene-specific riboprobes were detected with nanogold-anti-digoxigenin antibody conjugate. The CaM mRNAs were differentially distributed in both the neuronal and glial cell compartments. The greatest difference in neuronal distribution of the CaM mRNAs was found in the dendrites, where the mRNAs transcribed from the CaM I and III genes were much more abundant than the CaM II mRNA. The neuronal perikarya were heavy labeled for all the CaM mRNAs. Interestingly, the myelinated axons and axon terminals also contained small amounts of nanogold particles for all the CaM mRNAs, which diminished with increasing distance from the soma. Most of the synaptic profiles, however, contained labeling only in the postsynaptic region. The CaM mRNAs were differentially distributed in the glial cells. While the glial cell somata were only lightly labeled, surprisingly concentrated labeling was present in the perisynaptic and perivascular astrocytic processes. In general, the CaM II mRNA was the least represented in the glial processes. Only a very low CaM gene expression was observed in the endothelial and resting microglial cells. These results provide ultrastructural evidence for differential targeting of the multiple CaM mRNA transcripts to the intracellular compartments and suggest their microdomain-specific regulation.

Animals↗

PubMed2005

Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach.

The intragenic heterogeneity encountered in many dominant disease-causing genes represents a significant challenge with respect to development of economically viable therapeutics. For example, 25% of autosomal dominant retinitis pigmentosa is caused by over 100 different mutations within the gene encoding rhodopsin, each of which could require a unique gene therapy. We describe here an RNA interference (RNAi)-based mutation-independent approach, targeting as an example murine rhodopsin. Native transcripts are suppressed by a single RNAi molecular species, whereas transcripts from replacement genes engineered at degenerate third-codon wobble positions are resistant to suppression. We demonstrate suppression of murine rhodopsin transcript by up to 90% with full concomitant expression of replacement transcript and establish the validity of this approach in cell culture, retinal explants, and mouse liver in vivo.

Animals↗

PubMed2004

RNAi of COL1A1 in mesenchymal progenitor cells.

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Alleles↗

PubMed2003

Intracellular targeting of calmodulin mRNAs in primary hippocampal cells.

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.

Animals↗

PubMed2002

Differential calmodulin gene expression in the rodent brain.

Apparently redundant members of the calmodulin (CaM) gene family encode for the same amino acid sequence. CaM, a ubiquitous cytoplasmic calcium ion receptor, regulates the function of a variety of target molecules even in a single cell. Maintenance of the fidelity of the active CaM-target interactions in different compartments of the cell requires a rather complex control of the total cellular CaM pool comprising multiple levels of regulatory circuits. Among these mechanisms, it has long been proposed that a multigene family maximizes the regulatory potentials at the level of the gene expression. CaM genes are expressed at a particularly profound level in the mammalian central nervous system (CNS), especially in the highly polarized neurons. Thus, in the search for clear evidence of the suggested differential expression of the CaM genes, much of the research has been focused on the elements of the CNS. This review aims to give a comprehensive survey on the current understanding of this field at the level of the regulation of CaM mRNA transcription and distribution in the rodent brain. The results indicate that the CaM genes are indeed expressed in a gene-specific manner in the developing and adult brain under physiological conditions. To establish local CaM pools in distant intracellular compartments (dendrites and glial processes), local protein synthesis from differentially targeted mRNAs is also employed. Moreover, the CaM genes are controlled in a unique, gene-specific fashion when responding to certain external stimuli. Additionally, putative regulatory elements have been identified on the CaM genes and mRNAs.

Animals↗

PubMed2002

Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene.

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Amino Acid Sequence↗

PubMed2002

Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho(-/-) mice.

Comparative analysis of the transcriptional profiles of approximately 6000 genes in the retinas of wild-type mice with those carrying a targeted disruption of the rhodopsin gene was undertaken by microarray analysis. This revealed a series of transcripts, of which some were derived from genes known to map at retinopathy loci, levels of which were reduced or elevated in the retinas of Rho(-/-) mice lacking functional photoreceptors. The human homologue of one of these genes, encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), maps to the region of 7q to which an adRP gene (RP10) had previously been localized. Mutational screening of DNA from the Spanish adRP family, originally used to localize the RP10 gene, revealed an Arg224Pro substitution co-segregating with the disease phenotype. The amino acid at position 224 of the IMPDH1 protein is conserved among species and the substitution is not present in healthy, unrelated individuals of European origin. These data provide strong evidence that mutations within the IMPDH1 gene cause adRP, and validate approaches to mutation detection involving comparative analysis of global transcription profiles in normal and degenerating retinal tissues. Other genes showing significant alterations in expression include some with anti-apoptotic functions and many encoding components of the extracellular matrix or cytoskeleton, a possible reflection of a response by Muller cells to preserve the remaining outer nuclear layer of the retina. We suggest that those genes identified are prime candidates for etiological involvement in degenerative retinal disease.

Amino Acid Motifs↗