Gregory M. Ross

Multipotent neurotrophin antagonist targets brain-derived neurotrophic factor and nerve growth factor.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are members of the neurotrophin family that normally play a role in the development and maintenance of the nervous system. However, neurotrophin dysregulation has been implicated in several neurodegenerative diseases and psychiatric disorders including Alzheimers disease, Parkinsons disease, neuropathic pain, depression, and substance abuse. Despite their central role in the nervous system, neurotrophins have proved to be an elusive pharmacological target. Here, we describe a novel multipotent neurotrophin antagonist, 3-[(5E)-4-oxo-5-[[5-(4-sulfamoylphenyl)-2-furyl]methylene]-2-thioxo-thiazolidin-3-yl]propanoic acid (Y1036). Y1036 binds BDNF (KD = 3.5 ± 0.3 μM) and NGF (KD = 3.0 ± 0.4 μM) preventing either BDNF or NGF from interacting with their obligate receptor(s). Y1036 prevents both BDNF- and NGF-mediated trk activation, downstream activation of the p44/42 mitogen-activated protein kinase pathway, and neurotrophin-mediated differentiation of dorsal-root ganglion sensory neurons. Identification of a BDNF- and NGF-specific antagonist is of considerable interest in the study and treatment of diseases where dysregulation of multiple neurotrophins has been implicated.

Identification of novel catecholamine absorbing proteins in the central nervous system

AbstractSeveral pharmacologically active catecholamines have been shown to react covalently with CNS proteins, namely species of 47, 40, 22, and 20 kDa. Of these, the 47-kDa protein showed the greatest incorporation of tritium following treatment with [3H]dopamine, [3H]ADTN, or [3H]N-propyl-norapomorphine. Labeling was accomplished by incubating the tritiated ligands with crude membrane preparations in the absence of reducing agents. These proteins displayed several unique characteristics:1.The proteins are distributed throughout the CNS, but no evidence was found for their presence in other tissues;2.The proteins have a unique pharmacological profile, interacting with dopamine, ADTN, N-propylnorapomorphine, and apomorphine, but not with ligands specific for other proteins known to interact with these compounds;3.The labeling of these proteins is not inhibited by several similar catecholamines and other catechols, suggesting specific structural requirements; and4.These proteins exhibited stereoselectivity with respect to this labeling.n These results demonstrate the existence of novel CNS proteins capable of covalently absorbing several physiologically important catecholamines in vitro.

The binding of zinc and copper ions to nerve growth factor is differentially affected by pH: implications for cerebral acidosis

It has recently been shown that transition metal cations Zn2+ and Cu2+ bind to histidine residues of nerve growth factor (NGF) and other neurotrophins (a family of proteins important for neuronal survival) leading to their inactivation. Experimental data and theoretical considerations indicate that transition metal cations may destabilize the ionic form of histidine residues within proteins, thereby decreasing their pKa values. Because the release of transition metal cations and acidification of the local environment represent important events associated with brain injury, the ability of Zn2+ and Cu2+ to bind to neurotrophins in acidic conditions may alter neuronal death following stroke or as a result of traumatic injury. To test the hypothesis that metal ion binding to neurotrophins is influenced by pH, the effects of Zn2+ and Cu2+ on NGF conformation, receptor binding and NGF tyrosine kinase (trkA) receptor signal transduction were examined under conditions mimicking cerebral acidosis (pH range 5.5–7.4). The inhibitory effect of Zn2+ on biological activities of NGF is lost under acidic conditions. Conversely, the binding of Cu2+ to NGF is relatively independent of pH changes within the studied range. These data demonstrate that Cu2+ has greater binding affinity to NGF than Zn2+ at reduced pH, consistent with the higher affinity of Cu2+ for histidine residues. These findings suggest that cerebral acidosis associated with stroke or traumatic brain injury could neutralize the Zn2+‐mediated inactivation of NGF, whereas corresponding pH changes would have little or no influence on the inhibitory effects of Cu2+. The importance of His84 of NGF for transition metal cation binding is demonstrated, confirming the involvement of this residue in metal ion coordination.

Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene

Abstract: The recently described catecholamine‐absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high‐specific‐activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high‐specific‐activity substituted tetrahydronaphthalene derivative (6‐hydroxy‐[125I]iodo‐[N‐(N‐2′,4′‐dinitrophenyl)aminopropyl]‐2‐amino ‐ 1,2,3,4‐tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.

Characterization of High‐Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate‐Solubilized Bovine Striatal Preparations

Abstract 3,4‐Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid‐NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane‐bound receptors. Computer‐assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high‐ and low‐affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high‐affinity sites to low‐affinity sites for the agonists. The binding of the high‐affinity agonist [3H]N‐n‐propylnor‐apomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of gua‐nylyl‐imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl‐Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six‐ to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high‐affinity binding sites than the second peak. The solubilized preparation also showed high‐affinity binding of [35S]guanosine‐5′‐(γ‐thio)triphos‐phate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor‐guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor‐effector interactions.

Effects of unilateral intracerebroventricular microinjections of cholecystokinin (CCK) on circling behavior of rats

Unilateral intracerebroventricular (ICV) injections of a high dose of CCK7 have been reported to elicit barrel rotations accompanied by contralateral postural asymmetry; there was no spontaneous locomotor activity other than barrel rolling. The aim of the present study was to investigate the effects of lower doses of CCK-peptides on circling behavior; it was reasoned that if ambulation was present following unilateral ICV administrations of lower doses of CCK, then the contralateral postural asymmetry previously reported might be expressed as contraversive circling. In the present study, spontaneous locomotor activity was observed following ICV injections of lower doses of CCK sulfated octapeptide (CCK8), desulfated CCK octapeptide (dCCK8) and CCK tetrapeptide (CCK4). As postulated, contraversive circling was induced by CCK8 (0.5-5000 ng, ICV); the two other CCK fragments, dCCK8 and CCK4, were inactive in this respect. In addition, the contraversive circling bias induced by CCK8 (5.0 ng, ICV) was attenuated by co-injections of the CCK antagonist proglumide (10 and 100 ng) and by intraperitoneal injections of the dopamine (DA) antagonist haloperidol (0.05 and 0.1 mg/kg, 45 min prior to ICV CCK8). These data suggest that the effect is medited by CCK receptors and through a facilitatory influence on central DA function.

Factors affecting solubilization of the D1 receptor from bovine caudate nucleus

The D1 dopamine receptor was identified using the dopamine agonist 3H-ADTN. The ability of various detergents to solubilize this receptor from bovine caudate nucleus was examined. In order to maintain high affinity binding, receptors were solubilized in the presence of dopamine and manganese. GPP(NH)P reduced the yield of soluble receptors. Of the detergents tested, n-octyl-B-D-glucopyranoside gave the best yield of receptors capable of binding 3H-ADTN and related compounds with high affinity.

Sulfated cholecystokinin octapeptide (CCK8) failed to modulate basal or dopamine-stimulated adenylate cyclase activity in the rat striatum

1. In view of previously demonstrated modulatory effects of CCK8 on DA-sensitive adenylate cyclase activity in the nucleus accumbens, we examined the effects of this neuropeptide in the striatum. 2. Adenylate cyclase activity was measured by conversion of alpha-[32P]ATP into [32P]cAMP. 3. CCK8, when added to the adenylate cyclase assay in concentrations up to 100 microM, failed to significantly alter, either positively or negatively, basal or DA-stimulated (30 or 100 microM) adenylate cyclase activity. Similar results were obtained in the presence of various peptidase inhibitors. 4. Under the assay conditions employed in these experiments, it would appear that there is no effect of CCK8 on DA-sensitive adenylate cyclase in the striatum.