Acetylcholinesterase expression in NTera 2 human neuronal cells: a model for developmental expression in the nervous system.

Acetylcholinesterase (AChE; EC 3.1.1.7) is expressed in the central nervous system in multiple molecular forms that may subserve multiple functions and may be selectively lost in neurodegenerative illnesses such as Alzheimer's disease. AChE expression has been studied in primary cultures of developing vertebrate nervous system, but investigation has been limited by the lack of a suitable human CNS surrogate cell model system for in vitro studies and the inability of primary brain cultures to provide large numbers of pure neurons. To develop an in vitro model for studies of neuronal AChE expression and function, we utilized a neuronally committed human teratocarcinoma cell line, NTera 2, that can be induced to differentiate to a post-mitotic CNS neuronal phenotype. We found that NTera 2 cells express multiple molecular forms of AChE that are similar to CNS-derived AChE isoforms in velocity sedimentation profile, anion exchange elution profile, and sensitivity to inhibitors. At least two forms of AChE are expressed (G1 and G4), similar to human and rodent brain, and induction of NTera 2 cell differentiation results in an increased G4/G1 ratio, which is characteristic of mature neurons. As in primary CNS neurons, AChE is present in NTera 2 cells in both the cytosolic fraction and in the outer membrane, and is also released in a soluble form. These observations indicate that NTera 2 cells provide a useful human model system for studies of expression of cell-associated and soluble cell-free AChE in developing and mature human neurons and for elucidating the potential role(s) of acetylcholinesterase metabolism in both normal development and neurodegenerative disease states.

Human immunodeficiency virus type 1 Tat activity in human neuronal cells: uptake and trans-activation.

Neurological dysfunction in AIDS occurs in the absence of productive infection of neurons, and may involve modulation of neuronal cell function by viral or cellular products released from surrounding infected cells. The human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat may be one such factor, as it can act as a neurotoxin, induces marked morphological changes in neurons and astrocytes in primary embryonic rodent brain cultures, and is released by certain HIV-1-infected cells. In addition, Tat can alter expression of cellular genes in several non-neuronal cell types. To explore the possibility that Tat may also mediate neuronal dysfunction in AIDS through non-lethal effects on neurons, we determined the trans-activating ability of Tat in human neuronal cells. We generated human neuronal cell lines stably expressing several HIV-1 tat genes, and also tested human neuronal cells exposed to extracellular recombinant Tat protein. Both endogenously expressed Tat as well as exogenous recombinant Tat protein up-regulated HIV-1 long terminal region (LTR)-driven gene expression by several hundred-fold. Only brief exposure to recombinant Tat was necessary and no toxic effects were seen at levels sufficient for trans-activation. Furthermore, Tat significantly enhanced virus expression in neuronal cells transfected with molecular clones of HIV-1. These results show that Tat is trans-activationally active in human neuronal cells, and can be taken up from the extracellular compartment by these cells in a biologically active form. Neurons represent an important potential target for Tat-mediated cellular dysfunction.