Jane Talvenheimo

Immunocytochemical localization of TrkB in the central nervous system of the adult rat

The TrkB family of transmembrane proteins serve as receptors for brain‐derived neurotrophic factor (BDNF), neurotrophin (NT)‐4/5, and possibly NT‐3, three members of the neurotrophin family of neurotrophic factors. In order to understand the potential roles played by these receptors, we have examined the distribution of the TrkB receptor proteins in the adult rat brain by using immunohistochemistry. Several different antisera, directed against either synthetic peptides corresponding to different regions of TrkB or a recombinant fusion protein comprising part of the extracellular domain, were generated. Each of these antisera was directed to epitopes found on all known TrkB isoforms (both the tyrosine kinase‐possessing isoform and the truncated kinase‐lacking isoforms). In addition, a commercially available antibody to the intracellular domain of TrkB was also used. Widespread and distinct staining was observed on the surface of neuronal cell bodies, axons, and dendrites in many structures, including the cerebral cortex, hippocampus, dentate gyrus, striatum, septal nuclei, substantia nigra, cerebellar Purkinje cells, brainstem and spinal motor neurons, and brainstem sensory nuclei. Staining was also observed in the pia matter, on a subpopulation of ependymal cells lining the cerebral ventricle wall, and other nonneuronal cells. The expression pattern of TrkB receptor protein suggests that TrkB plays a broad role in the central nervous system. In addition, the detection of TrkB immunoreactivity on cell bodies and dendrites is consistent with recent models suggesting that neurotrophins may be derived from presynaptic and/or autocrine sources in addition to the classical postsynaptic target. J Comp Neurol 378:135–157, 1997. 1997

AMG 706, an Oral, Multikinase Inhibitor that Selectively Targets Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors, Potently Inhibits Angiogenesis and Induces Regression in Tumor Xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

Polyglutamine repeat length-dependent proteolysis of huntingtin.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntingtons disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation

Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC50 values for pH 5 and 5.5 were 364.68 ± 29.78 and 28.31 ± 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation.

Interactions between Brain-derived Neurotrophic Factor and the TRKB Receptor IDENTIFICATION OF TWO LIGAND BINDING DOMAINS IN SOLUBLE TRKB BY AFFINITY SEPARATION AND CHEMICAL CROSS-LINKING

The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF). Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF. One of these domains consists of amino acid residues 103–181 and contains both the third leucine-rich motif and the second cysteine cluster domain. The second domain is close to the second immunoglobulin-like domain (amino acid residues 342–394). Each of these two domains can bind BDNF independently. Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation. To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248–398) was expressed in Escherichia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction. Several synthetic peptides corresponding to amino acid residues 343–379 were able to bind immobilized BDNF. Amino acid substitution and cross-linking analysis indicated that amino acids Phe347, Asp354, and Tyr361 are intimately involved in BDNF binding. These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF.