Most patients with AIDS will develop a disabling dementia characterized by memory loss, blindness, and motor disturbances. Recent evidence indicates that this is not due to infection of neurons with HIV-1, but possibly to a neurotoxic effect of the viral coat protein gp120 shed from infected cells. The mechanism of gp120 action appears in some respects to be similar to normal mechanisms of glutamate activation, and excessive stimulation of neurons with excitatory amino acids (EAA) is also neurotoxic. The molecular mechanisms by which gp120 or EAA stimulation result in lethal cellular injury are unknown, although one pathway appears to involve post-transnational alterations in the neuronal spectrin cytoskeleton. The overall goal of the proposed studies will be to understand how the neuronal spectrin cytoskeleton organizes pre- and postsynaptic receptors, stabilizes the neuronal membrane, and is altered by gp120 and EAA action. Specifically, research will focus on the factors which sort unique isoforms of spectrin, ankyrin, and adducing to the pre-or postsynaptic membrane, and regulate the interactions between these proteins and with other components of the neuronal membrane. Neurons specific components of interest include a putative non CD4 gp120 receptor and the NMDA receptor. Regulatory events acting on the spectrin skeleton to be examined include u-calpain mediated proteolysis, Ca++ and calmodulin, and phosphorylation by cAMP dependent and independent kinases, calmodulin kinases, and protein kinase C. The distribution of unique isoforms and of post-translationally modified proteins are identified in normal and AIDS patient's brain by immunofluorescent and immuno-EM using monoclonal and sequence specific antibodies; interactions between purified or recombinant protein components are characterized by in vitro assay, their rate of incorporation into the neuronal cytoskeleton and post-transnational modification after EAA or recombinant gp120 stimulation are studied in cultured neuronal cells. Functional sites in spectrin critical for in vivo assembly at the neuronal membrane or for (functional) linkage to the gp120 or NMDA receptors will be identified by the ability of expressed recombinant wild type and mutated proteins to associate with the membrane in cultured neuronal cells, and be post-translationally modified following receptor activation.
| Malchiodi-Albedi, F; Ceccarini, M; Winkelmann, J C et al. (1993) The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specific domains of cerebellar neurons. J Cell Sci 106 ( Pt 1):67-78 |
