Françoise Boiron

Decreased biosynthesis of saturated C20-C24 fatty acids by the trembler mouse sciatic nerve.

Excised Trembler mouse sciatic nerves synthesize, from acetate, only minute amounts of C20 and C22 saturated fatty acids (about 1 10 of the normal value) and almost no lignoceric acid. The elongation activity is localized in the microsomal fraction. The microsomes from Trembler sciatic nerves can elongate stearoyl-CoA into C20, C22 and C24 saturated fatty acids. The elongation rate is only 1 3 of the normal value, whereas the stearoyl-CoA hydrolysis is 3 times higher than in the control; the malonyl-CoA concentration remains at the same level in microsomes from normal and trembler sciatic nerves. When ATP-Mg(2+) is added to the Trembler microsomes, the stearoyl-CoA hydrolysis is reduced, the stearoyl-CoA concentration remains nearly normal and the elongation reaches an almost normal level.

Evidence of an abnormal stearoyl-CoA hydrolysis in a membrane pellet from trembler sciatic nerves

Abstract A membrane fraction from sciatic nerves of Trembler mouse hydrolyzed stearoyl-CoA, 2–3 times more efficiently than the controls. The maximal hydrolysis within 60 min could reach 40% of the added substrate as compared to 20% in the control. Stearoyl-CoA hydrolysis increased as a function of time (up to 60 min), protein amount (up to 200 μg) and substrate concentration. Addition of ATP and Mg 2+ markedly lowered the free fatty acid release in the membrane pellet from Trembler mouse sciatic nerves.

Phospholipid acylation by mouse sciatic nerve microsomes

The partition of 0.3 nmol of [1-14C]oleoyl-CoA in the microsomes (10 micrograms proteins) from mouse sciatic nerves is unaffected by the presence of lysophospholipids and is about 45% of the total oleoyl-CoA (77% of the acylglycerophosphocholine partition in the membrane). The concentration of both oleoyl-CoA and acylglycerophosphocholine is over 1 mM in the membrane. There is a selective acyl transfer from acyl-CoA to lysolipid acceptors (oleoyl greater than myristoyl, palmitoyl, stearoyl much greater than eicosanoyl greater than docosanoyl, tetracosanoyl). The exogenous acyl acceptors are acylglycerophosphocholine and acylglycerophosphoinositol and to a lesser extent acylglycerophosphoethanolamine, but not acylglycerophosphoserine. A PC formation from acylGPC in the absence of exogenous acyl donors or from oleoyl-CoA in the absence of exogenous acyl acceptor was also observed.

Assay of stearoyl-CoA synthesis in microsomes from normal and Trembler mouse sciatic nerves.

Using a new one-step technique which allows the characterization and a quantitative assay of long-chain acyl coenzyme A (acyl-CoA), the activity of the stearoyl-CoA synthetase was studied in the membrane fraction from sciatic nerves of normal and Trembler mice. A synthesis of acyl-CoAs was observed in both cases, but, whatever the experimental conditions, there was a two-fold increase of the specific activity of the ligase in the PNS of the Trembler mouse.

Po, MBP, histone, and DNA levels in sciatic nerve

We studied the quantitative changes in proteins (total, Po, MBP, and histones) and DNA from sciatic nerves of normal and Trembler mice during postnatal development. Polyacrylamide gel electrophoresis and immunoblotting procedures allowed an accurate characterization of Po, MBP, and histones, as well as the comparison of their respective amounts from d 2 to d 120 after birth. It was found that 1. The immunoblotting procedure ascertains the presence of Po in the sciatic nerve of Trembler. In the 2-d-old mice, Po is detected in essentially similar amounts in Trembler and normal PNS, whereas its level in adult mutant sciatic nerves is never greater than 20% of the control. The sharp increase in Po levels observed during the third week in the normal nerves is not observed in those of the mutant; 2. MBP species are at most 4% of the control in the 10- to 12-d-old Trembler mice, whereas they were not detectable in adult nerves. The distribution of the different MBP species is the same in both mutant and control mice; 3. In normal mice, Po and MBP accumulate at similar rates, but the 14 kDa MBP accumulates faster than the 18.5 kDa MBP; and 4. Histone and DNA contents decrease 3- to 5-fold in normal nerves, whereas they remain constant, or increase slightly, in the mutant.

Technique for injection into the sciatic nerve of the mouse for quantitative in vivo metabolic studies

In this paper we describe a technique for intraneural injections, applicable to mouse peripheral nerves, which, compared with previous techniques, reduces trauma to the nerves and increases the level and reduces the variability of label recovery. Our technique employs glass needles (tip diameter, 50 micron) linked to a peristaltic pump by polyethylene tubing to inject small volumes (in the microliter range) of radiolabeled substrate solutions into mouse sciatic nerves, and allows the recovery of 20.9 +/- 1.9% (mean +/- standard deviation) and 30.5 +/- 4.8% of the injected radioactivity for 2 microliter [3H]acetate and 0.5 microliter of [3H]stearate, respectively.

A comparison of substrate-dependent fatty acyl-CoA synthetase activities and ATP hydrolysis in membrane pellets from normal and trembler mouse sciatic nerves.

Acyl-CoA synthetase activity was investigated in membrane fractions from normal and Trembler mouse sciatic nerves. Stearoyl-CoA synthesis in the Trembler assays was always about twice that observed in the control experiments under various conditions of CoA and ATP concentrations. In experiments using varying concentrations of fatty acid, the activation of stearate was very low in both systems (normal and Trembler), up to a substrate concentration of 20 mM, suggesting that fatty acid micelles are required for full activity. ATP, at concentrations of up to 1 mM, was totally (Trembler) or partially (control) hydrolyzed within 15 min, resulting in an apparently lower ligase activity in the mutant than in the control when an ATP-regenerating system was not added. The activation of saturated fatty acids decreased with increasing chain length. However, lignoceroyl-CoA formation was detected in the mutant but not in the control.

Evidence for an Intermembrane Transport of Lipids to Peripheral Nerve Myelin in vivo by a Novel Pulsed Wave Procedure

Three principal methods have been employed for the study of intracellular transport in nervous tissues: autoradiography associated with electron microscopy, time-staggered double-isotope labelling and isotopic pulse-chase experiments. Although the autoradiographical (Gould and Dawson, 1976; Gould et al., 1987; Kumara-Siri and Gould, 1980; Rawlins, 1973) and the double-labelling methods (Konat et al., 1985; Pereyra et al., 1983; Shimomura et al., 1984) can provide useful complementary information, only the isotopic pulse-chase experiments (Benjamins and Iwata, 1979; Burkart et al., 1982; Konat, 1981; Konat et al., 1985), associated with the use of intracellular transport inhibitors, would be able to demonstrate unambiguously the existence of intermembrane transfer events implicated in membrane biogenesis. No in vivo studies of peripheral nerves have been carried out using this method, most probably because of the difficulty in controlling the experimental conditions.