Failure of GPI compounds to display neurotrophic activity in vitro and in vivo.

The aim of this study was to evaluate the neurotrophic and neuroprotective properties of a series of immunophilin ligands and to assess the potential involvement of FK506 Binding Protein 12 kDa (FKBP12) rotamase inhibition in this activity. Both FK506 and rapamycin induced a potent inhibition of the FKBP12 rotamase activity (pIC(50) values of 7.3 and 7.4, respectively) but only a modest inhibition was observed with 1-(3,3-dimethyl-2-oxo-pentanoyl)-pyrrolidine-2-carboxylic acid S-3-pyridin-3-yl-propyl ester (GPI 1046) (5.8), its N-oxide (5.4) and thioester (6.3) analogues. Compared to nerve growth factor, all these immunophilin ligands only induced marginal increases in neurite outgrowth of rat dissociated newborn dorsal root ganglia cells. Furthermore, systemic administration of GPI 1046 and its N-oxide and thioester analogues failed to prevent striatal dopamine depletion induced by acute or chronic i.p. treatment with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP). These results suggest that inhibition of FKBP12 rotamase activity is not predictive for neurotrophic and neuroprotective properties of immunophilin ligands and question their therapeutic utility in neurodegenerative diseases like Parkinson's disease.

LCAT activation properties of apo A-I CNBr fragments and conversion of discoidal complexes into spherical particles.

We studied the substrate properties of the phospholipid-cholesterol-apolipoprotein complexes generated with apo A-I, apo A-I-CNBr fragments, apo A-II and apo A-IV for cholesterol esterification by the enzyme lecithin-cholesterol acyltransferase (LCAT). The kinetic parameters determined with the different complexes as substrates, showed that the complexes containing apo A-I and apo A-IV were about 40-times more efficient than those generated with the apo A-I fragments. In this system, the substrates containing apo A-II had the lowest efficiency. In spite of the differences in the kinetic parameters observed with the various apolipoprotein-lipid complexes, the cholesterol inserted in the complexes was esterified for more than 90% after 24 h in all systems studied. Based upon the results of the kinetic experiments, we followed the transformation of the discoidal complexes into spherical particles, due to the formation of a cholesteryl esters core, in the presence of low-density lipoproteins as an external source of cholesterol. We observed the formation of spherical particles by electron microscopy, after incubation of the discoidal complexes with LCAT for 24 h. The average percentage of cholesteryl esters in the converted particles was around 60% of the total cholesterol, varying between 40% for the apo A-I-CNBr-1-DPPC-cholesterol complex and up to 86% for the apo A-I-DPPC-cholesterol complex. The secondary structure of protein in the complexes was not significantly modified. However, the phospholipid phase transition disappeared, together with the parallel orientation of the phospholipid acyl chains with the helical segments of the apolipoproteins, as the phospholipids are organized in a monolayer at the surface of the spheres.

Characterization of the discoidal complexes formed between apoA-I-CNBr fragments and phosphatidylcholine.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.