The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.

The in vitro binding of a new subtype‐selective NMDA receptor antagonist, [3H]Ro 25‐6981, to rat brain membranes and sections was characterized. The compound bound to a single site on the membranes with a KD of 3 nM and a Bmax of 1.6 pmol/mg of protein. Specific binding, defined with a new NR2B‐specific antagonist, Ro 04‐5595 [1‐(4‐chlorophenyl)‐2‐methyl‐6‐methoxy‐7‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline], at 10 µM, was fully inhibited by several compounds with the following rank order of affinities—Ro 25‐6981 > CP‐101,606 > Ro 04‐5595 = ifenprodil ≫ eliprodil > haloperidol > spermine > spermidine > MgCl2 > CaCl2—and partially inhibited by competitive glutamate recognition site antagonists. A high density of binding sites was detected, radioautographically, in several layers of the cerebral cortex, in the hippocampus, dentate gyrus, tuberculum olfactorium, caudate putamen, medium densities in the globus pallidus, thalamus, spinal cord dorsal horn, and motoneurons, whereas the cerebellum, pons, and medulla were, with a few exceptions, e.g., locus coeruleus, poorly labeled. Overall, the distribution of [3H]Ro 25‐6981 binding sites correlated well with that of NR2B (but not NR2A) transcripts, revealed by in situ hybridization histochemistry. The high affinity of [3H]Ro 25‐6981 for NR2B‐containing receptors renders this compound the ligand of choice to study the regulation of NR2B‐containing receptor expression.

No abstract

Abstract:We have investigated the binding properties of [ 3 H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K D values of 27 Ϯ 4 and 81 Ϯ 22 nM for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S)-4-Carboxyphenylglycine, (S)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [ 3 H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM [ 3 H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 M kainate, revealing the distribution of mGlu1ϩ5 receptors. The latter were almost completely inhibited by the group I agonist (S)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively. Key Words: [ 3 H]Quisqualate binding-Metabotropic glutamate 1a and 5a receptors-Radioautography. J. Neurochem. 75, 2590 -2601 (2000).Eight G protein-coupled metabotropic glutamate (mGlu) receptors have been cloned to date (for review, see Pin et al., 1999). On the basis of their sequence similarities, signal transduction, and agonist rank order of potency, these receptors have been subdivided into three groups. Group I mGlu receptors, for which several splice variants have been identified , are activated by the weak group I-selective agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-3-hydroxyphenylglycine (3HPG) and the potent nonselective agonist quisqualate (Aramori and Nakanishi, 1992). This group comprises mGlu1, which is selectively blocked noncompetitively by 7-(hydroxyimino)cyclopropa [b]chromen1a-carboxylate ethyl ester (CPCCOEt) (Litschig et al., 1999), and mGlu5, which is selectively blocked, noncompetitively, with high potency by methylphenylethynylpyridine . Although precise physiological roles cannot yet be assigned to mGlu1 and mGlu5 receptors (potent, selective agonists/antagonists with good brain penetration are not yet widely available), group I receptors have, nevertheless, been implied in psychiatric and neurological diseases (see Bordi and Ugolini, 1999).

Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [(3)H]LY354740 ((+)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and -3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca(2+) and Mg(2+) ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740-stimulated [(35)S]GTPgammaS binding (25-97% above basal, according to the brain region), revealing G protein-coupled receptor coupling to G(i) proteins. Nonspecific binding (in the presence of 10 muM DCG-IV, a group II-selective, mGlu2-preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [(3)H]DCG-IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimer's disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al. [2004] Neuropharmacology 46:907-917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.