In vivo transcriptional regulation of the human immunodeficiency virus in the central nervous system in transgenic mice.

Human immunodeficiency virus type 1 (HIV-1) causes infections of the central nervous system (CNS) and has been implicated as the causative agent of AIDS-associated encephalopathy and the AIDS dementia complex. The development of in vivo models of HIV-1-mediated gene expression has shown that the HIV long terminal repeat (LTR) from the viral isolate HIV(JR-CSF) specifically supports gene expression in adult and developing CNS. To determine the molecular basis for HIV-1 developmental CNS gene expression, in vivo footprinting analysis by the ligation-mediated PCR technique was performed on CNS tissue from the brain stem of a transgenic mouse. The association of cellular proteins in the CNS with sequences in the LTR was found over sequences that defined the TATA region, the Sp-1 and NF-kappaB sites, and two upstream regions (-111 to -150 and -260 to -300). A purine-rich sequence at positions -256 to -296 of the HIV(JR-CSF) LTR but not of the HIV(IIIB) LTR specifically bound protein in nuclear extracts of newborn brain tested in electrophoretic mobility shift assays. No specific protein binding was observed to this region in liver or HeLa cell nuclear extracts. This suggests the presence of a newly identified transcription factor involved in regulation of HIV-1 gene expression in the CNS.

HIV-1 in the developing CNS: developmental differences in gene expression.

HIV-1 infection of the CNS plays a direct role in the pathogenesis of AIDS dementia that frequently accompanies systemic AIDS. Both adult and pediatric AIDS are characterized by a high proportion of CNS disease. However, the pathogenic mechanisms responsible for AIDS dementia are not understood. A transgenic mouse model using the LTRs of two CNS-derived strains of HIV-1 (HIV-1JR-CSF and HIV-1JR-FL) has been developed to study HIV-1 gene expression in vivo. Analyses of expression in adult transgenic mice revealed expression in neurons in the CNS (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clement, Science 258, 1804-1808, 1992). In this study, developmental analyses of HIV-1-directed gene expression in embryonic and newborn transgenic mice derived from the above lines revealed strikingly different levels and patterns of expression in the CNS and spinal cord compared with adult mice. Increased expression was observed in the newborn brain compared to the adult, and the neuroanatomical pattern of expression was markedly different than that observed in adult brain. Transient expression was detected in the dorsal root ganglia and spinal cord in embryos and newborns up to Day 14. In contrast to the expression in neurons in adult CNS, HIV-1-directed gene expression in the newborn brain was observed in neurons, endothelial cells, and macrophages. This difference in expression during development probably reflects temporally regulated cellular transcription factors in the CNS. This transgenic model suggests that HIV-1 replication in the CNS may use cellular transcription factors different from those in nonneural tissues. Studies are in progress to identify cellular transcription factors that may be responsible for the differential expression of the LTRs.

Expression directed from HIV long terminal repeats in the central nervous system of transgenic mice.

Infection with the human immunodeficiency virus (HIV) is frequently accompanied by the AIDS (acquired immunodeficiency syndrome) dementia complex. The role of specific HIV genetic elements in the pathogenesis of central nervous system (CNS) disease is not clear. Transgenic mice were constructed that contained the long terminal repeats (LTRs) of two CNS-derived strains and a T cell tropic strain of HIV-1. Only mice generated with CNS-derived LTRs directed expression in the CNS, particularly in neurons. Thus, some strains of HIV-1 have a selective advantage for gene expression in the brain, and neurons can supply the cellular factors necessary for their transcription.

Automated image analysis for counting unstained cultured neurones.

A fully automated image analysis technique was developed for counting the number of live or fixed, unstained neurones present in a representative region of a cell culture dish. A dish containing cultured mouse hippocampal neurones was placed on the motorized stage of an inverted microscope, and the neurones were visualized using Hoffman modulation contrast optics. The resulting image was digitized, and processed by subtracting the background illumination, low pass filtering, thresholding, then deleting objects whose areas fell outside a specified range. Two threshold levels were used, each with its own area range, and the two resulting binary images were combined. The number of objects in the combined image was counted. The number of cells in each field was also counted manually, and the processing was repeated on a series of 100 fields covering a representative region of the dish. The automated counts were highly correlated with the manual counts for each of the 12 culture dishes examined in this study. The correlation coefficient was calculated for the manual and automated counts from each dish, and the values ranged from 0.91 to 0.97. Six of the dishes were treated with the envelope protein of the human immunodeficiency virus (gp120), which reduces survival of neurons in this system. The six treated dishes were found to have significantly fewer neurones than the six control dishes, using either manual or automated counting techniques.

Identification of a domain in cytochrome C that displaces [3H]TCP binding from rat brain membrane receptors: synthesis of beta-neuroprotectin.

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue, [3H]TCP, was utilized to screen for inhibition of binding in extracts of rat brain. 2. Extracts were prepared from rat cerebral cortex and hippocampus by methods employing aqueous acid. The extracts were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The middle zone was further purified by high performance-size exclusion chromatography (HP-SEC). 3. Size exclusion chromatography revealed a single zone of activity corresponding to mol. wts of ca 12,000-31,000 daltons. A fraction from this zone was digested with trypsin, and the resulting enzyme fragments, isolated by a combination of HP-SEC and RR-HPLC, were identified as fragments of rat cytochrome C. 4. Horse cytochrome C was digested with trypsin and the fragments were similarly purified on the basis of the [3H]TCP binding displacement assay. The fragments were sequenced and found to be trypsin cleavage products of a single largely invariant domain of the cytochrome C molecule: Lys-Lys-Lys-Asp-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Lys. 5. beta-neuroprotectin (D)-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-NH2, inhibits [3H]TCP binding and provides protection against NMDA mediated neuronal cell death at low concentrations.