Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.

BACKGROUND: Aspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by gamma-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFkappaB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on gamma-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein. RESULTS: Exposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFkappaB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity. CONCLUSION: Nelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFkappaB and matrix metalloproteinase 2 in cerebral endothelial cells.

Altered P-glycoprotein expression in AIDS patients with HIV encephalitis.

Penetrance of anti-retroviral drugs into the CNS depends partly on the activity of P-glycoprotein (P-gp), an ATP-dependent efflux pump involved in restricting entry of lipophilic drugs into the brain. The present study characterizes the patterns of P-gp expression in the brains of AIDS patients and examines its relationship with clinical and neuropathological indicators of HIV encephalitis (HIVE). For this purpose, brain tissue collected at autopsy from 26 subjects with a history of HIV (9 without HIVE; 17 with HIVE) was analyzed. Immunocytochemical staining and Western blot analyses for regional P-gp expression were performed and levels were correlated with neuropathological indicators and with HIV RNA. Double labeling experiments were performed with antibodies against astroglial (GFAP), endothelial (CD31), microglial (CD45) and neuronal (MAP2) cell markers. In the HIVE-negative cases, P-gp immunoreactivity was associated primarily with endothelial cells. HIVE-positive cases showed extensive immunolabeling of astroglial and microglial cells, but relatively less endothelial cell immunolabeling. No neuronal P-gp immunostaining was detected in brain tissue from any cases in the study. In the HIVE-positive cases with extensive astroglial labeling, the most intense immunoreactivity was detected in white matter. A subset of HIVE-positive cases displayed intense P-gp immunostaining of astrocytes closely associated with blood vessels in the cortex. Both the immunocytochemical and Western blot analyses showed a significant correlation between P-gp expression and HIV RNA levels. In conclusion, P-gp immunoreactivity was detected largely in glial cells in tissue from HIVE-positive patients. Furthermore, in HIVE-positive patients, brain viral burden and P-gp levels were significantly higher than those in HIVE-negative patients. Taken together, our data suggest that P-gp may be part of a central pathway mediating viral compartmentalization in the brains of HIV-infected individuals and may play a significant part in HIV disease progression in the brain.

The role of mitochondrial alterations in the combined toxic effects of human immunodeficiency virus Tat protein and methamphetamine on calbindin positive-neurons.

The use of methamphetamine (METH) continues to increase the risk of human immunodeficiency virus (HIV) transmission within both homosexual and heterosexual drug abuser groups. Neurological studies indicate that the progression of HIV encephalitis is also enhanced by illicit drug use. Recently, the authors' studies in the postmortem brains of HIV-positive METH users have shown that the combined effects of HIV and METH selectively damage calbindin (CB)-immunoreactive nonpyramidal neurons, which may contribute to the behavioral alterations observed in these patients. To better understand the mechanisms of toxicity associated with exposure to HIV and METH, neuronal survival, phenotypic markers, levels of oxidative stress, and mitochondrial potential were assessed in vitro in the hippocampal neuronal cell line, HT22, and in primary human neurons exposed to the HIV Tat protein and/or METH. Both Tat and METH were toxic to neurons in a time- and dose-dependent fashion. Neurons exposed to a combination of Tat and METH displayed early evidence of neuronal damage at 6 h, characterized by a decrease in CB and microtubule-associated protein 2 (MAP2) immunoreactivity followed by more extensive cell death at 24 h. Loss of CB immunoreactivity associated with the combined exposure to Tat and METH was accompanied by mitochondrial damage with increased levels of oxidative stress. The toxic effects of Tat and METH were inhibited by blocking mitochondrial uptake of intracellular calcium, whereas blocking calcium flux in the endoplasmic reticulum or from the extracellular environment had no effect on Tat and METH toxicity. These studies indicate that in vitro, when combined, the HIV protein Tat and METH damage CB-immunoreactive nonpyramidal neurons by dysregulating the mitochondrial calcium potential. In combination, Tat and METH may increase cell injury and death, thereby enhancing brain metabolic disturbances observed in HIV-positive METH users in clinical populations.

Patterns of selective neuronal damage in methamphetamine-user AIDS patients.

The risk for HIV infection attributable to methamphetamine (METH) use continues to increase. The combined effect of HIV and METH in the pathogenesis of HIV encephalitis (HIVE) is unclear, however. To better understand the neuropathology associated with HIV and METH use, the patterns of neurodegeneration were assessed in HIV-positive METH users and in HIV-positive non-METH users. Patients in the study met criteria for inclusion and received neuromedical and postmortem neuropathologic examinations. Immunocytochemical and polymerase chain reaction analyses were performed to determine brain HIV levels and to exclude the presence of other viruses. METH-using patients with HIVE showed significantly lower gp41 scores and less severe forms of encephalitis but a higher frequency of ischemic events, a more pronounced loss of synaptophysin immunoreactivity, and a more severe microglial reaction than HIVE non-METH users. Furthermore, in METH-using patients with HIVE, extensive loss of calbindin (CB)-immunoreactive interneurons displaying phylopodial neuritic processes suggestive of aberrant sprouting was observed. Taken together, these studies indicate that the combined effects of METH and HIV selectively damage CB immunoreactive nonpyramidal neurons. In combination, METH and HIV may increase neuronal cell injury and death, thereby enhancing brain metabolic disturbances observed in clinical populations of HIV-positive METH abusers.

Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability.

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.