Background: Astrocytes maintain physiologic concentrations of the excitatory neurotransmitter glutamate in the CNS. Glutamate excess disrupts synaptic signaling and neuronal circuitry. Infection with HIV-1 or exposure to HIV-1 proteins may impair glutamate uptake or metabolism in astrocytes, contributing to HIV dementia. Although astrocytes selectively cultured from primary human fetal brain cells (HFBC) have been used to study these processes, a novel human CNS progenitor-derived astrocyte developed in this lab is highly susceptible to HIV-1 clone pNL4-3 transfection, suggesting that progenitor-derived astrocytes might also be used to examine HIV-1 neuropathophysiology. This study first compared glutamate uptake and expression of GLT-1 and GLAST transporters in GFAP (+) HFBC and progenitor-derived astrocytes, then measured the effect of TNF-alpha and gp120 incubation upon glutamate uptake in progenitor-derived cells.

Methods: GLT-1 and GLAST expression were determined by immunohistochemistry, Western blot and FACS. (3H)glutamate uptake was measured by liquid scintillation, expressed as counts/min/microgram protein. (3H)glutamate uptake after 24 hr incubation with TNF-alpha (20ng), gp120 (200pM) individually and then in combination was determined in progenitor-derived astrocytes.

Results: Immunohistochemistry and FACS were positive for the presence of GLT-1 and GLAST in both cell lines. Both were also positive for the presence of GLT-1 by Western blot, but negative for GLAST using a commercially available antibody. HFBC and progenitor-derived astrocytes exhibit biphasic (3H)glutamate uptake. Progenitor-derived astrocytes incubated with either TNF-alpha or gp120 alone failed to change glutamate uptake compared to untreated cells, whereas concurrent TNF-alpha and gp120 exposure decreased glutamate uptake compared to controls.

Conclusions: GFAP (+) HFBC and human progenitor-derived astrocytes express glutamate transporters GLT-1 and GLAST, with biphasic uptake of (3H)glutamate. Concurrent 24 hr incubation of progenitor-derived astrocytes with TNF-alpha (20ng) and gp120 (200pM) reduced glutamate uptake, perhaps reflecting a cooperative effect. These preliminary findings, combined with susceptibility to HIV-1 transfection, suggest that astrocytes derived from a novel multi-potential human progenitor cell line may be used to examine glutamate transport and metabolism under conditions of HIV-1 infection and inflammation.