The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 12s51_ t384;6 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 ,uM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. It is now established that infection with human immunodeficiency virus type 1 (HIV-1) is often complicated by neurological syndromes that include dementia, subacute encephalitis, and vacuolar degeneration of the spinal cord (9,19,28,32). The identification and isolation of HIV-1 from the brain suggests that the retrovirus itself is responsible for the neurological disorders observed in HIV-infected patients. Other lentiviruses, including visna virus (15) and simian immunodeficiency virus (22), are also associated with brain infections. Among central nervous system (CNS) cells, monocyte and macrophage lines are preferentially infected by HIV, but infection of other neural cell types has also been discussed (20,35).The pathogenic mechanism by which the virus causes encephalopathy remains unknown. It was reported recently that the HIV envelope glycoprotein manifests neurotoxic activity by increasing free Ca2+ in rat neurons, thus causing cellular damage (4, 7). This effect can be prevented by Ca2+ channel antagonists.As an approach to another possible cause of neurological dysfunction, we investigated whether other HIV proteins could be implicated in this pathology. For this study, numerous peptides were chemically synthesized on an Applied Biosystems peptide synthesizer (model 430A) with the stepwise solid-phase method (25,29).By testing the neurotoxicity of synthetic fragments of various lengths, derived from gp160, p25, nef, and tat proteins, we discovered that the intracerebroventricular injection of tat or some tat fragments caused toxic and lethal effects in mice. The 86-residue tat protein from HIV-1 has been previously reported to be critical for virus replication through its role in viral trans activation (1,11,12,14,18,33 We have further investigated tat neurotoxicity by structure-activity relationships, using binding experiments and electrophysiology. We first investigated the capacity of radiolabeled tat3886 from HIV-1, LAVBru isolate, to bind to rat brain synaptic nerve ending particles (synaptosomal membranes) prepared by the method of Gray and Whittaker (13). Protein was measured by a modified Lowry method (24). 125I-tat38 86 (>10-8 M), the most active peptide in vivo (Table 1), bound to...

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 12s51_ t384;6 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 ,uM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.

The plant-derived insecticides have introduced a new concept in insecticide research. In response to insect attacks, some plants can release volatile sulfur compounds such as dimethyl disulfide (DMDS) in the atmosphere, which are lethal for the generalist insects. We demonstrate that DMDS induced an uncommon complex neurotoxic activity. The studies of in vivo toxicity of DMDS in three insect species and mice indicated a highest bioactivity for insects. Although DMDS did not alter the electrophysiological properties of the cockroach Periplaneta americana giant axon, it affected the synaptic transmission at the presynaptic level resulting in an inhibition of the neurotransmitter release. Whole cell patch-clamp experiments performed on cockroach cultured dorsal unpaired median (DUM) neurons revealed a dose-dependent hyperpolarization induced by DMDS associated with a decrease in the input resistance and the disappearance of action potentials. The hyperpolarization was inhibited by glibenclamide and tolbutamide, and was dependent on intracellular ATP concentration, demonstrating a neurotoxicity via the activation of KATP channels. Finally, the same effects observed with oligomycin, 2,4-dinitrophenol, and KCN together with the studies of DMDS toxicity on isolated mitochondria confirmed an unusual action occurring through an inhibition of the mitochondrial respiratory chain complex IV (cytochrome oxydase). This DMDS-induced inhibition of complex IV subsequently decreased the intracellular ATP concentration, which thereby activated neuronal KATP channels mediating membrane hyperpolarization and reduction of neuronal activity.

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