Klotho Is a Neuroprotective and Cognition-Enhancing Protein.

In this chapter, we will describe what has been learned about Klotho and its potential functions in the brain. Klotho is localized in the choroid plexus and, to a lesser extent, in hippocampal neurons. Cognitive decline is a common issue in human aging affecting over 50% of the population. This cognitive decline can also be seen in animal models such as the Rhesus monkey. A long-term study undertaken by our lab demonstrated that normal brain aging in rhesus monkeys and other animal models is associated with a significant downregulation of Klotho expression. This observation substantiates data from other laboratories that have reported that loss of Klotho accelerates the development of aging-like phenotypes, including cognitive deficits, whereas Klotho overexpression extends life span and enhances cognition in mice and humans. Klotho is a type 1 transmembrane pleiotropic protein predominantly expressed in kidney and brain and shed by ADAM 10 and 17 into the blood and cerebral spinal fluid, respectively. While the renal functions of Klotho are well known, its roles in the brain remain to be fully elucidated. We recently demonstrated that Klotho protects hippocampal neurons from amyloid and glutamate toxicity via the activation of an antioxidant enzymatic system suggesting Klotho is a neuroprotective protein. Furthermore, Klotho is necessary for oligodendrocyte maturation and myelin integrity. Through its diverse roles in the brain, Klotho has become a new therapeutic target for neurodegenerative diseases such as Alzheimer's disease and demyelinating diseases like multiple sclerosis. Discovery of small molecule Klotho enhancers may lead to novel treatments for these incurable disorders.

Quinine- and quinidine platelet antibodies can react with GPIIb/IIIa.

Quinine- and quinidine-dependent antiplatelet antibodies are generally believed to bind to the membrane glycoprotein complex, GPIb/IX. However, we and others have found that some drug-dependent antibodies bind to platelets from patients with the Bernard-Soulier syndrome which lack these glycoproteins. We therefore studied the reactions of a group of these antibodies with normal and Bernard-Soulier platelets and their membrane proteins. As indicated by rosette formation of the sensitized platelets around protein A-Sepharose beads, two quinine- and two quinidine-dependent antibodies reacted with both normal and Bernard-Soulier syndrome platelets at a high (300 microM) concentration of drug. At a pharmacologic drug concentration (10 microM), all four antibodies reacted with normal platelets but only the two quinine-induced antibodies reacted with Bernard-Soulier platelets. Immunoprecipitation studies with solubilized, tritium-labelled normal platelets, at both high and low drug concentrations, showed that each of the four antibodies precipitated proteins corresponding to GPIb and GPIX. Fainter bands corresponding to glycoproteins IIb and IIIa, which do not label well with tritium, were also detected. With radioiodinated normal platelets, it was found that each of the four antibodies was capable of precipitating GPIIb/IIIa, but only in the presence of drug. The four antibodies also promoted drug-dependent precipitation of GPIIb and GPIIIa from lysates of radioiodinated Bernard-Soulier platelets. The two quinine-dependent antibodies precipitated these glycoproteins at both high and low drug concentrations, while the quinidine-dependent antibodies reacted much more strongly at the higher drug concentration. Precipitation of GPIb/IX was not observed with BSS platelets. Absorption of a quinine-induced antibody with Bernard-Soulier platelets in the presence of drug eliminated its ability to precipitate GPIIb and GPIIIa. However, the absorbed antibody retained the ability to precipitate GPIb from solubilized normal platelets. Thus, at least two drug-dependent antibodies were present, one specific for GPIb/IX and the other for GPIIb/IIIa. These findings indicate that glycoproteins IIb and/or IIIa, in addition to the GPIb/IX complex, can serve as targets for drug-dependent antibodies in both intact and detergent-solubilized platelet preparations.

Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F(ab')2 fragments of goat antibody specific for the Fc portion of human IgG, while rosette formation between antibody-coated protein A-Sepharose and platelets was inhibited by F(ab')2 fragments directed against the F(ab')2 portion of the IgG molecule. Since binding of IgG to protein A is known to occur via the Fc region, these findings suggest that binding of drug-induced antibodies to platelets occurs at the Fab domains of the IgG molecule.

Structural features of the quinidine and quinine molecules necessary for binding of drug-induced antibodies to human platelets.

Although most quinidine- and quinine-induced platelet antibodies react only in the presence of the drug that provoked sensitization, some are active with either quinidine or its stereoisomer, quinine; that is, they are "cross-reactive." This suggests that the activity of drug-dependent antibodies is dependent on different structural features of the quinidine or quinine molecules. To investigate this possibility, we studied reactions of 16 quinidine- and quinine-induced antibodies with quinidine, quinine, and analogues of these drugs modified at the quinoline ring (desmethoxy-derivative), the quinuclidine ring (dihydro-derivative), or the asymmetric C(9)-hydroxyl position. It was found that the antibodies could be classified into three groups on the basis of their reactions with platelets in the presence of these compounds. Eight antibodies (group 1) reacted only in the presence of the sensitizing drug or its dihydro- or desmethoxy-derivative. Three antibodies (group 2) differed from those in group 1 only in that their reactions were markedly weakened when the primary desmethoxy-derivative was used. Five antibodies (group 3) reacted in the presence of the sensitizing drug, its stereoisomer, and one or more of the analogues tested, including at least one of the C(9)-derivatives. Antibodies in this group gave stronger reactions with the sensitizing drug than with its stereoisomer. These results provide further evidence for heterogeneity among drug-induced platelet antibodies by demonstrating that noncross-reactive antibodies (groups 1 and 2) are dependent for their activity on a specific configuration at the optically active C(9)-hydroxyl position, and that some of these (group 2) also require the methoxy group for full reactivity. In contrast, cross-reactive antibodies (group 3) appear to be dependent on the quinoline ring common to all the analogues tested but also require a specific configuration at C(9) for full reactivity.