Bernard Croizat

PARTICIPATION OF THE MITOCHONDRIAL GENOME IN THE DIFFERENTIATION OF NEUROBLASTOMA CELLS

SummaryUsing 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.

Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord--human muscle cocultures.

Neurotrophins, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and brain-derived neurotrophic factor (BDNF), were studied in vitro in a coculture model of human skeletal muscle myotubes and rat embryo spinal cord explants, which enables the different steps of functional innervation to be followed, including neurite outgrowth, synapse formation and induction of contractile activity. We found that NT-3, NT-5, BDNF, but not NGF simultaneously induced a significant increase in the number and length of neurites emerging from spinal cord explants, the number of endplates per muscle fiber, and the area of innervated muscle fibers around each spinal cord explant. These results suggest that neurotrophins NT-3, NT-5 and BDNF enhance spinal cord motoneurons potential of innervation.

Regulation of Peripherin in Mouse Neuroblastoma and Rat PC 12 Pheochromocytoma Cell Lines

Peripherin (Formerly the Y protein) is found in the peripheral nervous system. This Triton-insoluble protein is characterized by its isoelectric point (5.6), its apparent molecular weight (56,000 daltons) and its peptide map. Peripherin was also observed in a mouse neuroblastoma cell line, NIE 115, where its expression appeared regulated by the presence of an inducer of morphological differentiation. In order to analyze more precisely this control, the presence of peripherin was investigated in several neuroblastoma cell lines which exhibit different morphological patterns of differentiation and in the rat pheochromocytoma PC 12 cell line. Differentiation of these cells was induced with 1-methylcyclohexane carboxylic acid (CCA) and nerve growth factor (NGF), respectively. Peripherin was found in these different cell lines. Moreover, the cellular amount of peripherin appraised by [35S]-methionine incorporation was significatively increased in differentiated cells. In contrast, other cytoskeletal components did not undergo a similar raise. The level at which the control of the peripherin content takes place was studied in a cell-free translation system. Poly(A)-rich RNAs extracted from growing or differentiated NIE 115 cells directed the synthesis of similar amounts of peripherin in a reticulocyte lysate. In contrast, polysomes prepared from differentiated cells and the corresponding polysomal RNA programmed in vitro the synthesis of twice more peripherin than polysomes or polysomal RNA from growth-phase cells. Since peripherin synthesis is enhanced 5 times in living cells, it seems probable that the cellular amount of peripherin is controlled partly at the translational level and partly at the turn-over level.

Mitochondrial maturation during neuronal differentiation in vivo and in vitro

Summary— The evolution of the mitochondrion has been followed within differentiating neuronal cells, both in primary cultures of neurons from fetal rat cortex and during rat brain cortex maturation. Changes in total mitochondrial proteins (mt‐proteins) were evaluated, and qualitative changes in the mt‐proteins pattern were analyzed using the Western blot technique. The evolution of mt‐protein contents in cultured neurons resembles what is observed during rat brain maturation. The mitochondrion exhibits pronounced changes in the course of neurogenesis, in particular, bursts of mitochondrial masses accompanying the successive steps of neurogenesis are observed. There are indications that protein equipment of mitochondria during neuronal development undergoes variations. Although more work is required to establish the significance of these correlations, the present data might suggest an important role of the mitochondrion in neurogenesis.

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.

Degeneration of cocultures of spinal muscular atrophy muscle cells and rat spinal cord explants is not due to secreted factors and cannot be prevented by neurotrophins

We have shown recently that cocultures of muscle cells from infantile spinal muscular atrophy (SMA) patients innervated by motoneurons of normal rat spinal cord explants undergo a degeneration process, suggesting that muscle may play a role in this atrophy, which previously has been considered to be a pure motoneuron disease. Conditional media of SMA cocultures did not affect control healthy nerve muscle cocultures. Conversely, conditioned media of control cocultures were unable to prevent degeneration of SMA cocultures. Moreover, neurotrophic factors, thought to be of help in motoneuron disease treatment, did not protect SMA cocultures from premature death. Our results suggest that the abnormal phenotype observed in nerve‐muscle coculture (1) is not due to the release of a toxic factor nor to the lack of a secreted survival factor; and (2) does not respond to neurotrophin treatment.

Effects on the cytoskeleton of a new inducer of the neuroblastoma morphological differentiation

Abstract 1-Methyl cyclohexane carboxylic acid (CCA), a new inducer of neuroblastoma morphological differentiation, was found to stimulate the synthesis of vimentin in neuroblastoma cultures. Synthesis of actin and isotubulins was also modified, thus implying a more general effect on the cytoskeleton. Most of these variations are observed in culture conditions allowing interactions between the cell membrane and a solid substrate.

Complexity of polysomal polyadenylated RNA in mouse whole brain and cortex

Data concerning the eukaryotic messenger sequence complexities broadly differ according to the techniques of measurement. The analysis of hybridization kinetics of cDNA to mRNA template tends to underestimate the complexity as compared to saturation hybrid~ation of single copy DNA. This discrepancy has been considerably emphasized in the case of mouse brain poly~A)RNA. The data range from w 10 000 sequences [ 1 ,l’@J to 100 000 f 121. Causes for this discrepancy are not totally explained, however, by the difference in the technical approaches, since important variations also appear in the results reported by authors using the same technique. For instance, using cDNA, -19 000 sequences were found [2] as compared to 11 600 [ 11. In many cases, these variations may be explained, at least partly, by differences in the experimental protocols or (and) by the use of two different analytical methods (computer analysis or linear plot) for obta~i~g best fit to the data. Independently of the technical approach, and its obvious impact on the quantitative conclusions, a higher sequence complexity has been claimed [2-51 for mouse brain than for other tissues. These authors generally interpret the high level of poly(A)RNA sequence complexity as reflecting the high heterogeneity of cell types constituting the brain. The possibility has not been totally excluded, however, that a neuronal cell per se, perhaps because of its very large potential in estabB~g homotypic and hetero~p~c contacts, would express more genes than cells from any other tissues.

Tissue-specific mitochondrial proteins

Mitochondrial proteins from rat brain cortex, muscle, liver, and from neuronal cells in culture were compared on 2-D electrophoregrams. This analysis permitted characterization of certain specificities in the distribution of polypeptides depending on tissue localization. In particular, 16 mit-proteins were found exclusively in the mitochondrion from brain tissue.

Changes in the β-subunit of mitochondrial F1ATPase during neurogenesis

Abstract A polypeptide migrating in the area of the isotubulin in 2 D - gel electrophoresis of extracts from neuronal cells was characterized as the β-subunit of the F1ATPase matrix component. The synthesis of this subunit is enhanced during neurogenesis and the presence of an isoform was detected in adult mouse brain.