Use of a simple fractionation method to evaluate binding, internalization and intracellular distribution of oligonucleotides in vascular smooth muscle cells.

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4 degrees C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25 kDa polyethylenimine (PEI) as a carrier. After 2.5 h of incubation at 37 degrees C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A large proportion (about 60%) of intracellular radioactivity remained associated with the endocytic compartment. Complexation with PEI completely changed intracellular distributions: the nuclear fraction increased to 70% for PS-ODN. The fractionation method proposed, facilitating study of the subcellular distribution of the ODN, could also be used under appropriate circumstances, to study variations in cytosolic ODN content.

Giant axon and neurofilament accumulation in Charcot-Marie-Tooth disease type 2E.

The axonal type 2 Charcot-Marie-Tooth disease (CMT2) is phenotypically poorly characterized. Here the authors report a family with a Pro22Ser mutation in the neurofilament-light gene (NF-L; CMT2E) manifesting electrophysiologically as the demyelinating type 1 CMT (CMT1) and pathologically as an axonopathy with giant axons and accumulation of disorganized NF. NF-L should be investigated in CMT2 as well as in CMT1 not associated with the usual genes PMP22, Cx32, and P0.

Segmental conduction abnormalities and myelin thickenings in Val102/fs null mutation of MPZ gene.

The authors report in patients with Val102/fs null mutation a possibly age dependent variability of clinical and electrophysiologic phenotype, segmental conduction abnormalities mainly in ulnar nerves at the elbow, and excessive myelin foldings and thickenings. The authors hypothesize that myelin thickenings at the paranodal region, in concurrence with compression at usual entrapment sites or minor repetitive trauma, may induce segmental conduction abnormalities.