Perinatal maturation of rat kidney mitochondria.

In the rat kidney, NaK-ATPase activity increased between days 19 and 20 of gestation (+50%) and between 1 and 24 h after birth (+20%), requiring an increased energy supply. In order to determine whether mitochondrial changes were involved, renal mitochondrial development was investigated from day 19 of gestation to 1 day after birth. Slot-blot analyses of mitochondrial-DNA/nuclear-DNA ratio and determination of citrate synthase activity showed a doubling in the mitochondrial pool between days 19 and 20 of gestation. In isolated mitochondria, oxygen consumption remained unchanged between days 19 and 20 of gestation, and then it was enhanced between days 20 and 21 of gestation (+70%) and between 1 and 24 h after birth (+50%). We also focused on one of the respiratory-chain complexes, ATP synthase, and measured its activity and content during the perinatal period. We demonstrated increases in both activity and content of ATP synthase between days 20 and 21 of gestation and between 1 and 24 h after birth, thus suggesting that changes in ATP synthase activity are ascribed to an increase in the mitochondrial density of ATP synthase complexes. Moreover, the mitochondrial ATP/ADP ratio only increased between 1 and 24 h (+90%), indicating a critical step in the renal respiratory-chain maturation at that time. We therefore conclude that the postnatal enhancement of renal mitochondrial oxidative capacity might depend on protein synthesis de novo and on changes in the adenine nucleotide concentrations.

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

Adriamycin promotes neurite outgrowth in the "neurite-minus" N1A-103 mouse neuroblastoma cell line.

Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.

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.

Regulation of c- and N-myc expression during induced differentiation of murine neuroblastoma cells.

Using clones N1E-115 and N1A-103 from mouse neuroblastoma C1300, a comparative analysis of c- and N-myc gene expression was undertaken both in proliferating cells and in cultures exposed to conditions which induce differentiation. Under the latter conditions, while N1E-115 cells extend abundant neurites and express many biochemical features of mature neurons, clone N1A-103 stops dividing and expresses certain neurospecific markers but is unable to differentiate morphologically. In both clones, chemical agents, i.e. 1-methyl cyclohexane carboxylic acid (CCA) or dimethyl sulfoxide (DMSO), induce a decrease in c-myc expression. Similar results were found for N-myc gene in N1E-115 cells, but in contrast, in clone N1A-103, N-myc expression is increased with CCA and not modified with DMSO. Globally, this study favours the hypothesis that changes in c-myc expression would correspond to cell division blockade and differentiation, while modulations in N-myc are more closely related to an early phase of terminal differentiation.

Mitochondrial maturation during neuronal differentiation in vivo and in vitro.

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.