Background: HIV infection of the central nervous system frequently causes neurological disorders. To understand mechanisms that underlie neurodegeneration in HIV infection, novel cell culture models were developed.

Methods: Primary human microglia, astrocytes, and neurons were purified from human brain cell cultures based on differential expression of specific cell surface antigens selected by screening > 80 monoclonal antibodies by FACS analysis and immunohistochemistry. Cultures of defined cell populations were established and used for studies on HIV-induced neuronal apoptosis.

Results: Purified primary human neurons (>98%) were GFAP^-MAP2⁺NCAM-L1⁺, and exhibited typical neuronal morphology after 5 days in culture, whereas astrocytes were GFAP⁺MAP2^-NCAM-L1^-.  Glutamate and KCl induced Ca⁺⁺ signaling in purified neurons but not astrocytes. CXCR4, but not CD4 or CCR5, was detected on purified neurons (10-30%) and astrocytes (>90%). In the presence of soluble CD4 (sCD4), X4 HXB gp120 but not R5 YU2 or ADA gp120 bound to purified neurons and astrocytes in a dose-dependent manner.  In the absence of sCD4, there was negligible binding of HXB, YU2, or ADA gp120 to neurons and astrocytes. In cultures containing neurons, astrocytes and microglia, HXB gp120 induced higher levels of neuronal apoptosis than ADA gp120. The presence of sCD4 had no effect on gp120-induced neuronal apoptosis. CXCR4⁺ neurons, which increased gradually over time, were more susceptible to gp120-induced apoptosis than CXCR4^- neurons. Studies using purified neurons cultured in the presence or absence of astrocytes and/or microglia demonstrated that microglia are required for gp120-induced neuronal apoptosis. In contrast, astrocytes but not microglia were required for TNF-a- or IL-1b-induced neuronal apoptosis.

Conclusions: These findings suggest that HIV gp120 induces neuronal apoptosis primarily through its interaction with microglia, whereas TNF-a or IL-1b induces neuronal apoptosis through an astrocyte-dependent pathway. The development of these novel cell culture systems will facilitate studies on the complex interactions of neurons, astrocytes, and microglia that lead to neuronal dysfuction and cell death during HIV-1 neuropathogenesis.

Keywords: neuropathogenesis; gp120; microglia