Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

Calcium-evoked dendritic exocytosis in cultured hippocampal neurons. Part II: mediation by calcium/calmodulin-dependent protein kinase II.

Calcium-evoked dendritic exocytosis (CEDE), demonstrated in cultured hippocampal neurons, is a novel mechanism that could play a role in synaptic plasticity. A number of forms of neuronal plasticity are thought to be mediated by calcium/calmodulin-dependent protein kinase II (CaMKII). Here, we investigate the role of CaMKII in CEDE. We find that the developmental time course of CEDE parallels the expression of alphaCaMKII, a dominant subunit of CaMKII. An inhibitor of this enzyme, KN-62, blocks CEDE. Furthermore, 7 d in vitro neurons (which normally do not express alphaCaMKII nor show CEDE) can undergo CEDE when infected with a recombinant virus producing alphaCaMKII. Expression of a constitutively active CaMKII produces dendritic exocytosis in the absence of calcium stimulus, and this exocytosis is blocked by nocodazole, an inhibitor of microtubule polymerization that also blocks CEDE. These results indicate that CEDE is mediated by the activation of CaMKII, consistent with the view that CEDE plays a role in synaptic plasticity.

Vaccinia virus transfection of hippocampal slice neurons.

Here we describe a technique that uses a recombinant vaccinia virus to transfect neurons in rat hippocampal slices. This technique allows the use of molecular biological manipulations on neuronal tissue while maintaining intact synaptic function. This method should be useful in testing specific hypotheses regarding the role of synaptic proteins.

Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins.

Vaccinia virus can be used to infect cells in the CNS of frogs, Xenopus laevis, and Rana pipiens, both in vivo and in vitro. In vivo infections were accomplished by injection of viral solution into the tectal ventricle of stage 40-48 tadpoles or by local injections into distinct neural regions. Infections with high titer of virus injected into the ventricle resulted in the majority of cells in the brain expressing foreign protein, while cells in the retina and optic nerve showed no expression. Infection with lower viral titers resulted in fewer infected cells that were distributed throughout the otherwise normal tissue. Intense expression of foreign protein in the brain was observed 36 hr after injection and remained high for at least 4 days. Infected animals developed normally and had the same number of cells in the optic tectum as control animals. Infection with a recombinant virus carrying the gene for Green Fluorescent Protein labels neurons, so that infected cells can be observed in vivo. Vaccinia virus provides a versatile means to alter proteins in distinct populations of neurons in amphibia.