Involvement of intronic sequences in cell-specific expression of the peripherin gene.

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.

Transcriptional activation of the mouse peripherin gene by leukemia inhibitory factor: involvement of STAT proteins.

Peripherin is a neuron-specific intermediate filament protein whose expression is activated in vitro by the neuropoietic cytokines leukemia inhibitory factor (LIF) and interleukin-6. We have studied the mechanisms of transcriptional activation of the peripherin gene by LIF. In particular, we have identified a 70-bp element [peripherin cytokine-responsive element (Pe-CyRE)] within the 5'-flanking sequences of the mouse peripherin gene (between -930 and -860) that enhances transcription in two neuroblastoma cell lines, NBFL and LA-N-2, in response to LIF treatment. We have also shown by DNA mobility shift assays that treatment of cells by LIF induces the binding of protein complexes composed of at least two members of the signal transducers and activators of transcription (STAT) factor family to a cis element (Pe-APRE2) within Pe-CyRE. Furthermore, the entire Pe-CyRE, as well as Pe-APRE2, conferred responsiveness onto a heterologous thymidine kinase promoter. However, the response amplitude of the heterologous promoter to LIF was lower than that observed with the 5'-flanking sequences of the peripherin promoter, suggesting that cooperative interactions with surrounding sequences of the peripherin gene are required for a full transcriptional activation.

Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells.

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.

Aggregation of a subpopulation of vimentin filaments in cultured human skin fibroblasts derived from patients with giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a generalized disorder of intermediate filament networks which results in the formation of an ovoid aggregate in a large variety of cell types. We investigated the cytoskeletal organization of cultured skin fibroblasts derived from three GAN patients by indirect immunofluorescence, confocal, and electron microscopy. Whereas the organization of microfilaments seemed normal, the microtubule network appeared disorganized and tangled. The organization of the intermediate filament network, composed of vimentin, was probed with three antibodies directed against different epitopes: two vimentin-specific antibodies, a monoclonal antibody (mAb V9) and a polyclonal antibody, and a serum specific for all type III IFPs (PI serum). These experiments showed that 20% of cultured skin fibroblasts from GAN patients have a vimentin aggregate composed of densely packed filaments which coexists with a well-organized vimentin network. After depolymerization of microtubules with nocodazole, all fibroblasts from GAN patients contained a vimentin aggregate which seemed to arise from a subpopulation of vimentin filaments normally integrated in the vimentin network. Such aggregates were never observed in any condition in control fibroblasts. Moreover, the ultrastructural analysis of GAN cells revealed the presence of swollen mitochondria. We suggest that GAN may be due to a defect in a factor which stabilizes cytoplasmic intermediate filament networks, and we speculate on its identification and properties.

[Changes of some proteins expression of the spinal cord and muscles in infantile spinal muscular atrophy]. / Variations de l'expression de certaines protéines de la moelle épinière et du muscle dans l'amyotrophie spinale infantile.

The various types of childhood spinal muscular atrophy (SMA) represent a spectrum of clinical disorders resulting from the degeneration of motor neurons (MN). The genetic defect has been recently localized to chromosome 5q in the region 11.2-13.3. Under normal conditions, half of the motor neurons die during embryonic development, while the remaining 50% survive to innervate muscle fibers and form neuromuscular junctions. Numerous studies using in vivo and in vitro models have shown that survival of MNs depends on the presence of trophic factors of neuronal and muscular origin. However, at the present time, no molecular mechanisms can be proposed to account for the nature and the sequence of the interactions leading to the formation and maintenance of a functional neuromuscular junction. To gain a better understanding of the SMA disorders, an alternative to genetic studies consists in analyzing the molecular mechanisms underlying this pathology. Variations in the expression of proteins, for instance, might reflect the pathological phenotype. We thought it possible to detect differences in the protein(s) which would correlate with the molecular deficit of childhood SMA. We, therefore, compared the patterns of human protein expression from normal controls and SMA spinal cord and muscle. Significant variations in the expression of some proteins, which have been quantified by a computerized Bio-Image electrophoresis system, have been found. In particular, two proteins, a and b (126 kDa and 112 kDa) which are very probably common to spinal cord and muscle show a marked increase of their expression in children with SMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of the mitochondrial genome in the differentiation of neuroblastoma cells.

Using clonal cell lines isolated from murine neuroblastoma C1300, we investigated the mitochondrial changes related to neuronal differentiation and, more generally, the role played by the mitochondrion in this phenomenon. By different approaches (measurement of the mitochondrial mass, immunoquantification of specific mitochondrial proteins, or incorporation of Rhodamine 123), the differentiation of the inducible clone, N1E-115, was found associated with an important increase of the cellular content in mitochondria. This increase could be observed with differentiating N1E-115 cells maintained in suspension, i.e. under conditions where neurite outgrowth is prevented but other early stages of (biochemical) differentiation continue to occur. That these mitochondrial changes are likely to be correlated with these stages of neuronal differentiation, rather than with simple progression to the postmitotic stage, stems from comparative experiments with clone N1A-103, a neuroblastoma cell line variant that becomes postmitotic after induction but fails to differentiate and shows no modification in its cellular content in mitochondria. In accordance with these observations, chloramphenicol prevents differentiation when added together with the inducer. This effect is probably related to the inhibition of mitochondrial translation rather than to modification of the bioenergetic needs because oligomycine, a potent inhibitor of the mitochondrial ATP synthetase, shows no effect on neurogenesis. As a working hypothesis and in keeping with independently published models, we postulate that products resulting from mitochondrial translation could be involved in the organization of the cytoskeleton or of certain membrane components whose rearrangements should be the prerequisite or the correlates to early stages of neuronal differentiation.

Growth inhibition of N1E-115 mouse neuroblastoma cells by c-myc or N-myc antisense oligodeoxynucleotides causes limited differentiation but is not coupled to neurite formation.

Antisense oligodeoxynucleotides were found to be stable in the culture medium containing fetal calf serum (heat-inactivated 30 minutes at 65 degrees C) and in cells. Antisense oligomer treatment causes cessation of mitoses, but does not lead to morphological differentiation. Under antisense conditions, we have observed an increase in the amount of two neurospecific protein, namely peripherin and gamma-enolase. Comparison of the results obtained with chemical inducers and antisense oligodeoxynucleotides allows us to postulate three phases in N1E-115 differentiation: the first correspond to the arrest of mitosis, the second to the expression of a limited neuronal program, and the third to the morphological and electrophysiological differentiation.