Characterization of organotypic ventral mesencephalic cultures from embryonic mice and protection against MPP toxicity by GDNF.

We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP(+)) and glial derived neurotrophic factor (GDNF) to validate this novel culture model. Dopamine production and dopaminergic neuron soma size increased dramatically with time in vitro, whereas the number of dopamine neurons declined by approximately 30% between week 1 and week 2, which was further reduced after week 4. GDNF treatment (100 ng/mL) increased dopaminergic neuron soma size (up to 43%) and DOPAC production (approximately three-fold), but not the number of dopamine neurons in control cultures. One-week-old cultures were more vulnerable to MPP(+), than three-week-old cultures. The EC(50) for dopamine depletion after 2 days exposure and 15 days of recovery were 0.6 and 7 microm, respectively. Both pre-treatment and post-treatment with GDNF are important to obtain maximal protection against MPP(+) toxicity. In one-week-old cultures (5 microm MPP(+), 2 days) GDNF provided potent neuroprotection with dopamine contents reaching control levels and number of tyrosine hydroxylase (TH)(+) cells up to 80% of control, but in three-week-old cultures (10 microm MPP(+), 2 days) the protective potential of GDNF was markedly reduced. Long recovery periods after MPP(+) exposure are required to distinguish between reversible or irreversible toxic and/or trophic effects.

Association of Vpu-binding protein with microtubules and Vpu-dependent redistribution of HIV-1 Gag protein.

The efficient exit of HIV-1 particles from cells requires the action of the viral encoded protein Vpu. Vpu-binding protein (Ubp) is a cellular protein that interacts with both Vpu and the major structural component of the viral capsid (Gag) and appears to affect the efficiency of particle exit. Elucidation of the function of Ubp and characterization of the spatial distribution of Ubp may provide information pertinent to understanding the role of Ubp in virus replication. To investigate the subcellular location of Ubp, and to see whether Vpu affects the intracellular distribution of Gag, we carried out immunofluorescence localization in conjunction with confocal microscopy. Based on this analysis Ubp is present in both the nucleus and the cytoplasm. In the cytoplasm, Ubp appeared to be associated with microtubules as evidenced by cofluorescence with tubulin in the absence and in the presence of colchicine. However, cytoskeletal isolation and detergent extraction of cells resulted in association of Ubp with the soluble fractions, indicating that Ubp is not in tight association with microtubules. Moreover, flotation gradient analysis demonstrated that Ubp is cytoplasmic and not stably associated with the plasma membrane. Interestingly, expression of Vpu in cells resulted in redistribution of both Ubp and Gag to a location near the periphery of the cell. The effect of Vpu on both Ubp and Gag protein has implications for Vpu-mediated particle exit from cells.