Addition of the beta-hydroxy-beta-methylglutaryl-CoA (HmG-CoA) reductase inhibitor lovastatin to human HEK cells transfected with the amyloid precursor protein (APP) reduces intracellular cholesterol/protein ratios by 50%, and markedly inhibits beta-secretase cleavage of newly-synthesized APP. Exogenous water-solubilized cholesterol at 200 microg/ml concentration increases newly synthesized beta-amyloidogenic products four-fold. These intracellular changes are detectable by immunoprecipitation and immunofluorescent labelling. Analyses of the fragments captured from culture medium by an N-terminal anti-beta-amyloid antibody on ProteinChip arrays and detected using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry revealed that culture with cholesterol (200 microg/ml) increased secretion of beta-amyloid 1-40 by 1.8-fold, and increased secretion of beta-amyloid 1-42. Changes in APP processing by cholesterol may mediate the way in which the ApoE4 allele increases risk of developing Alzheimer's disease (AD) in western populations.

Neuroinflammatory diseases, such as multiple sclerosis (MS), result from aberrant leucocyte traffic into the central nervous system (CNS). To breach the specialized blood-brain barrier, activated leucocytes interact with CNS endothelial cells (EC) and activate a CD54-mediated signaling pathway controlling the Rho GTPase. To function correctly Rho requires posttranslational prenylation, and this can be inhibited by depleting the supply of isoprenoids through inhibition of the cholesterol synthesis pathway with HMG-CoA reductase inhibitors (statins). Here we show that treatment of brain EC in vitro with lovastatin inhibits Rho-mediated transendothelial T cell migration. This effect can be reversed by supplementation with mevalonolactone, the downstream product of HMG-CoA reductase, or by ectopic expression of myristoylated Rho, which remains active in the absence of prenylation. In a relapsing-remitting mouse model of MS, lovastatin treatment inhibited leucocyte migration into the CNS and significantly attenuated the development of both acute and relapsing clinical disease. These studies demonstrate that the indirect pharmacological inhibition of Rho proteins in brain EC by statins can inhibit a key stage in the pathogenesis of neuroinflammation, namely leucocyte migration across the blood-brain barrier. Theses studies demonstrate a novel effect of statins in modulating the immune response in neuroinflammtory diseases and may provide additional rationale for their use in the treatment of MS.

In order to examine the role of the ICAM-1 C-terminal domain in mediating transendothelial T-lymphocyte migration and ICAM-1 mediated signal transduction. Mutant human ICAM-1 molecules were expressed in rat brain microvascular endothelial cells. Expression of human wild type ICAM-1 in rat brain endothelial cells (EC) resulted in a significant increase over basal levels in both adhesion and transendothelial migration of rat T-lymphocytes. EC expressing ICAM-1, in which the conserved tyrosine residue at codon 512 was substituted with phenylalanine (hICAM-1Y512F), also exhibited increased lymphocyte migration albeit less than with wild type ICAM-1. Conversely, expression of truncated human ICAM-1 proteins, in which either the intracellular domain was deleted (hICAM-1ΔC) or both the intracellular and transmembrane domains were deleted through construction of a glycophospholipid anchor (GPI-hICAM-1), did not result in an increase in lymphocyte adhesion and their ability to increase transendothelial migration was attenuated. Truncated ICAM-1 proteins were also unable to induce ICAM-1 mediated Rho GTPase activation. Rat EC treated with cell permeant penetratin-ICAM-1 peptides comprising human or rat ICAM-1 intracellular domain sequences inhibited transendothelial lymphocyte migration but not adhesion. Peptides containing a phosphotyrosine residue were equally potent at inhibiting lymphocyte migration. These data demonstrate that the intracellular domain of ICAM-1 is essential for transendothelial migration of lymphocytes, and that peptidomimetics of the ICAM-1 intracellular domain can also inhibit this process. Such competitive inhibition of transendothelial lymphocyte migration, in the absence of an affect on adhesion, further implicates ICAM-1-mediated signalling events in the facilitation of T-lymphocyte migration across brain EC. Thus agents which mimic the ICAM-1 intracellular domain may be attractive targets for novel anti-inflammatory therapeutics.

The ICAM-1 mediated brain endothelial cell signalling pathway induced by adherent lymphocytes is a central element in facilitating lymphocyte migration through the tight endothelial barrier of the brain. Rho proteins, which must undergo post-translational prenylation to be functionally active, have been shown to be an essential component of this signalling cascade. In this study we have evaluated the effect of inhibiting protein prenylation in brain endothelial cells on their ability to support T lymphocyte migration. Endothelial cells treated in vitro with protein prenylation inhibitors (PTI) resulted in a significant reduction in transendothelial T lymphocyte migration. To determine the therapeutic potential of this approach an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, was induced in Biozzi ABH mice. Animals treated prior to disease onset with PTI exhibited a dramatic and significant reduction in both leucocyte infiltration into the central nervous system (CNS) and clinical presentation of disease compared to untreated animals. These studies demonstrate, for the first time, the potential for pharmacologically targeting CNS endothelial cell signalling responses, and particularly endothelial Rho proteins, as a means of attenuating leucocyte recruitment to the CNS.