Recombinant nef HIV-IIIB protein is toxic to human neurons in culture

The expression of HIV-1 negative factor (nef) has been positively correlate d with HIV disease progression [Z. Hanna, D.G. Kay, N. Rebai, A. Guimond, S , Jothy, P. Jocicoeur, Nef harbors a maker determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95 (1998) 1 63-175]. Nef expression has been detected in HIV infected human brains with neuronal damage [A. Ranki, M. Nyberg, V. Ovod, R/I. Haltia, I. Elovaara, R . Raininko, H. Haapsalo, K. Krohn, Abundant expression of HIV Nef and Rev p roteins in brain astrocytes in associated with dementia, AIDS 9(9) (1995) 1 001-1008; Y. Saito, L.R. Sharer, M.G. Epstein, J. Michaels, M. Mintz, M. Le nder, K. Golding, B.M. Blumberg, Overexpression of nef as a marker for rest ricted HIV-1 infection of astrocytes in postmorten paediatric central tissu es, Neurology 14 (1994) 474-480]. It is postulated that nef may contribute to the neuronal damage observed in the brain of those with late HIV disease . To test this, the potential toxicity of recombinant nef (from HIV-1 IIIB) was compared to the neurotoxin human tumour necrosis alpha (TNF alpha) on human brain cells in culture. SK-N-SH neuroblastoma, primary human neurons and glial cells were exposed to recombinant nef or TNF alpha protein for 3 days or twice over 6 days. Cell viability was assessed by Trypan Blue, lact ate dehydrogenase (LDH) release and MTT assays. Nuclear fragmentation was d etected using the Hoechst Blue nuclear dye assay, Both nef and TNF alpha (1 00 ng/ml) caused a significant 30% reduction of SK-N-SH cell numbers after 3 days exposure (P=0.001). At this time, exposure to nef caused evident fra gmented nuclei in these cultures. Human neuronal cultures had a 32 and 33% decrease in cell number after 6 days exposure to either nef or TNF alpha, r espectively (P<0.001). Furthermore, as previously shown [J. He, C.M. DeCast ro, G.R. Vandenbark, J. Busciglio, D. Gabuzda, Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protoonocogene, Proc. Natl. Acad. Sci . 94 (1997) 3954-3959], a 3-day exposure to nef significantly reduced human glial cell number by 25% (P=0.001). Recombinant nef and TNF alpha compromi se human neurons in culture. Thus, like other virotoxins, it is shown for t he first time that nef may also contribute to neuronal damage that has been reported in dementia in late HIV disease. (C) 2000 Elsevier Science B.V. A ll rights reserved.