To determine whether the presenilin 1 (PS1), presenilin 2 (PS2) and amyloid beta-protein precursor (APP) mutations linked to familial Alzheimer's disease (FAD) increase the extracellular concentration of amyloid beta-protein (A beta) ending at A beta 42(43) in vivo, we performed a blinded comparison of plasma A beta levels in carriers of these mutations and controls. A beta 1-42(43) was elevated in plasma from subjects with FAD-linked PS1 (P < 0.0001), PS2N1411 (P = 0.009), APPK670N,M671L (P < 0.0001), and APPV7171 (one subject) mutations. A beta ending at A beta 42(43) was also significantly elevated in fibroblast media from subjects with PS1 (P < 0.0001) or PS2 (P = 0.03) mutations. These findings indicate that the FAD-linked mutations may all cause Alzhelmer's disease by increasing the extracellular concentration of A beta 42(43), thereby fostering cerebral deposition of this highly amyloidogenic peptide.

No abstract

Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

The earliest event so far known that occurs in the brain affected with Alzheimer's disease (AD) is the deposition and fibril formation of amyloid beta-protein (A beta). A beta is cleaved from a glycosylated membrane protein, called beta-amyloid protein precursor, and normally secreted into the extracellular space. Here we report on the presence of membrane-bound A beta that tightly binds GM1 ganglioside. This suggests that this novel A beta species, rather than secreted A beta, may act as a 'seed' for amyloid and further that intracellular abnormalities in the membrane recycling already exist at the stage of amyloidogenesis.

Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid beta protein (A beta) deposited in the brains of patients with Alzheimer's disease (AD). To quantitate all A beta s present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of A beta. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all A beta (96%) ended at A beta 42(43). The other 18 AD brains contained increasing amounts of A beta ending at A beta 40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of A beta ending at A beta 42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of A beta ending at A beta 40 (76% of total A beta) was also present. Immunocytochemical analysis with monoclonal antibodies selective for A beta s ending at A beta 42(43) or A beta 40 confirmed that, in brains with minimal congophilic angiopathy, virtually all A beta is A beta ending at A beta 42(43) and showed that this A beta is deposited in senile plaques of all types. In the remaining AD brains, A beta 42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of A beta ending at A beta 40 were deposited, primarily in blood vessel walls, where some A beta ending at A beta 42(43) was also present. These observations indicate that A beta s ending at A beta 42(43), which are a minor component of the A beta in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.

No abstract

GPR40, one of the G protein-coupled receptors predominantly expressed in pancreatic β-cells, mediates enhancement of glucose-stimulated insulin secretion by free fatty acids. A potent and selective GPR40 agonist is theorized to be a safe and effective antidiabetic drug with little or no risk of hypoglycemia. Cyclization of the phenylpropanoic acid moiety of lead compound 1 produced fused phenylalkanoic acids with favorable in vitro agonist activities and pharmacokinetic profiles. Further optimization led to the discovery of dihydrobenzofuran derivative 9a ([(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate, TAK-875) as a potent, selective, and orally bioavailable GPR40 agonist, with a pharmacokinetic profile enabling long-acting drug efficacy. Compound 9a showed potent plasma glucose-lowering action and insulinotropic action during an oral glucose tolerance test in female Wistar fatty rats with impaired glucose tolerance. Compound 9a is currently in clinical trials for the treatment of type 2 diabetes mellitus.