Human apolipoprotein E4 accelerates beta-amyloid deposition in APPsw transgenic mouse brain.

The human apolipoprotein E4 (ApoE4) isoform is associated with genetic risk for Alzheimer's disease. To assess the effects of different ApoE isoforms on amyloid plaque formation, human ApoE3 and ApoE4 were expressed in the brains of transgenic mice under the control of the human transferrin promoter. Mice were crossed with transgenic mice expressing human amyloid precursor protein containing the Swedish mutation (APPsw), which facilitates amyloid beta peptide (A beta) production. The following progeny were selected for characterization: APPsw+/- x ApoE3+/- and APPsw+/-, APPsw+/- x ApoE4+/- and APPsw+/- littermates. All mice analyzed were wild type for the endogenous mouse APP and ApoE genes. Mice expressing ApoE4 in combination with APPsw have accelerated A beta deposition in the brain as assessed by enzyme immunoassay for A beta40 and A beta42 extractable in 70% formic acid, by assessment of amyloid plaque formation using thioflavin-S staining, and by immunohistochemical staining with antibodies specific for A beta40 or A beta42 and the 4G8 monoclonal or 162 polyclonal antibody. No difference in the rate of A beta deposition in the brain was seen in mice expressing ApoE3 in combination with APPsw. Thus, our data are consistent with the observation in Alzheimer's disease that ApoE4 is associated with increased accumulation of A beta in the brain relative to ApoE3.

Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity.

Mutations in the gene encoding the amyloid protein precursor (APP) cause autosomal dominant Alzheimer's disease. Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate its cleavage. Here we identify a new membrane-bound aspartyl protease (Asp2) with beta-secretase activity. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) receptors but with the opposite functionality.

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA(A) receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. 2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response. 3. These functional characteristics were monitored with Cl- currents measurements in cloned rat alphaxbeta2gamma2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA(A) receptors with binding of [35S]-TBPS, which is a high affinity ligand specific for GABA(A) receptors with exquisite sensitivity to allosteric modulations. 4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

Ibuprofen: effect on inducible nitric oxide synthase.

Treatment with ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDS) has been reported to decrease the incidence as well as slow down the progression of Alzheimer's disease. Understanding the mechanism of this therapeutic effect would provide a target for development of drugs which may be devoid of side effects observed with NSAIDs. In addition to inhibiting cyclooxygenase (COX), the NSAIDs have recently been shown to decrease inducible nitric oxide synthase (iNOS) activity. Ibuprofen and other NSAIDs had no direct effect on catalytic activity of iNOS, but decreased levels of iNOS mRNA. The mechanism of action of ibuprofen on reduction of iNOS activity has been further investigated in the present study using rat primary cerebellar glial cell cultures. In addition, the effect of ibuprofen on COX mRNA expression and prostaglandin formation was also studied. Glial cells treated with E. coli lipopolysaccharide (LPS) and interferony (INFgamma) for 16 h expressed iNOS and COX. Ibuprofen did not directly inhibit iNOS activity. However, when ibuprofen was incubated at the same time with LPS and INFgamma for 16 h, enzyme activity was reduced, with an IC50 of 0.76 mM. Ibuprofen concentration-dependently decreased iNOS mRNA levels, with an IC50 > 2 mM. Thus, there was no correlation between decrease in iNOS activity and reduction in iNOS mRNA levels. Ibuprofen decreased iNOS protein levels, as determined by Western blot, with an IC50 of 0.89 mM. The data suggest that the reduction in iNOS activity by ibuprofen is due to inhibition of post-transcriptional processing of this enzyme. Ibuprofen had no effect on constitutive COX (COX-1) or inducible COX (COX-2) mRNA expression. However, ibuprofen inhibited PGE2 formation with an IC50 of 0.86 mM. The anti-inflammatory actions of ibuprofen have been related to inhibition of COX and, subsequently, reducing prostaglandin formation. Since the potency of ibuprofen for inhibition of PGE2 formation and reduction in iNOS activity are similar, it is suggested that, at therapeutically effective doses, a decrease in iNOS activity may also occur in vivo. Therefore, reduction in iNOS protein levels in the brain may have a role in preserving the integrity of neurons in individuals susceptible to Alzheimer's disease.

U-19451A: a selective inducible nitric oxide synthase inhibitor.

Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock. Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures. Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus gamma-interferon were used for iNOS activity. Rat cerebellum and primary cerebellar granule cell cultures were utilized for neuronal NOS (nNOS) activity. S-methylthiourea (SMT) and L-nitroarginine methyl ester (L-NAME), selective iNOS and nNOS inhibitors, respectively, were chosen as standards. Both U-19451A and SMT were 4-times more selective for iNOS as compared to nNOS in tissues. U-19451A was more selective than SMT for iNOS inhibition using cultures. L-NAME was 16-31 times more selective for inhibiting nNOS activity. Based on the selectivity of U-19451A for iNOS inhibition, this drug would be expected to be effective in the treatment of diseases with inflammatory pathology without producing side effects associated with nNOS inhibition.

Dopamine D4 versus D2 receptor selectivity of dopamine receptor antagonists: possible therapeutic implications.

The dopamine D4 receptor, which is considered a close variant of the dopamine D2 receptor, has recently been cloned. Receptor binding studies demonstrated that clozapine, which is an effective antipsychotic agent but atypical in that it lacks the usual side effects of other antipsychotic agents, has high selectivity for the dopamine D4 receptor versus the dopamine D2 receptor. Comparative binding affinity studies have been carried out for a number of interesting dopaminergic agents using membranes prepared from cloned dopamine D2 and D4 receptor containing cells. It was found that clozapine is selective for the dopamine D4 vs. the D2 receptor by a factor of 2.8. Other compounds with dopamine D4 receptor selectivity were (+)-apomorphine (8.7), (+)-N-propyl-norapomorphine (NPA) (2.4) and melperone (1.3). Compounds with considerable selectivity for the dopamine D2 receptor were haloperidol (0.31), chlorpromazine (0.084), trifluoperazine (0.034) and raclopride (0.001). Overall, the results with the antipsychotic agents tested, support the concept that dopamine D4 receptor selectivity may confer clozapine-like antipsychotic efficacy and furthermore that dopamine D2 receptor selectivity may confer side effect liability (extrapyramidal side effects and tardive dyskinesia).