Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.

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 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.