Showing papers by "Jon Lindstrom published in 2009"

Anti-inflammatory effect of lifestyle changes in the Finnish Diabetes Prevention Study

Abstract: Aims/hypothesis Subclinical inflammation confers an increased risk of type 2 diabetes, cardiovascular disease, neurodegenerative disorders and other age-related chronic diseases. Physical activity and diet can attenuate systemic immune activation, but it is not known which individual components of a comprehensive lifestyle intervention are most effective in targeting subclinical inflammation.

Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity.

Abstract: The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of α1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human α1 subunits with α7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop α1 67–76 in combination with the N-terminal α helix α1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to α1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same α1 MIR sequences substituted was recognized by a majority of human, feline, and canine myasthenia gravis sera. The presence of the α1 MIR sequences in α1/α7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR.

Nicotine Activates and Up-Regulates Nicotinic Acetylcholine Receptors in Bronchial Epithelial Cells

Abstract: Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

UBXD4, a UBX-Containing Protein, Regulates the Cell Surface Number and Stability of α3-Containing Nicotinic Acetylcholine Receptors

Abstract: Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

Nicotinic acetylcholine receptor subunits in rhesus monkey retina.

Abstract: Acetylcholine (ACh) activates both nicotinic and muscarinic acetylcholine receptors (AChRs). The nicotinic AChRs (nAChRs) are ligand-gated cation channels and consist of pentameric complexes either composed of subunits α2-α6 and β2-β4 as α/β combinations or composed of subunits α7-α10 as homomeric or heteromeric structures.1–3 Two broad classes of nAChRs are recognized in brain. One class consists of heteromeric nAChR subtypes comprised of the α2-α6 and β2-β4 units with high agonist affinity but insensitivity to the snake toxin α-bungarotoxin (αBgt); the second class consists of homomeric (i.e., α7, α8, or α9) or heteromeric pentamers (i.e., combined α7, α8, α9, or α10 subunits) with lower agonist affinity but with high sensitivity to αBgt.3,4 The subunit compositions of nAChRs, which govern their pharmacological and functional properties, vary in different regions of the nervous system. In the mammalian retina, the cholinergic cells comprise two populations of amacrine cells, with somata in the inner nuclear layer (INL) or ganglion cell layer (GCL) respectively.5,6 The dendrites of the cholinergic INL cells stratify as a narrow band in the outer portion of the inner plexiform layer (IPL), and those of the cholinergic cells in the GCL stratify as a narrow band in the inner portion of the IPL. Functionally OFF cells, the cholinergic cells in the INL release ACh at the cessation or decrement of light stimulation5,6; functionally ON cells, the cholinergic cells in the GCL release ACh at light onset or increment.7 Ach, acetylcholinesterase inhibitors, or other cholinergic agents affect the response properties of many types of ganglion cells, including ON- and OFF-center ganglion cells and all motion sensitive ganglion cells.5,8–12 Based on electrophysiology, many of the retinal actions of ACh are mediated by nAChRs.8,13 That the application of ACh also decreases responses in the optic nerve likely reflects its summed effect on the activity of various ganglion cells.13 In rabbit retina, for instance, both αBgt-sensitive and αBgt-insensitive nAChRs modulate the light responses of subsets of ganglion cell types, including directionally selective ganglion cells as well as many subsets of brisk transient and brisk sustained ganglion cells.14–18 Both AChR classes are expressed by some ganglion cells, and there is evidence that nAChRs are expressed by upstream cells as well. For example, many ganglion cells and several types of amacrine cells express αBgt-insensitive β2-containing nAChRs, some of which are in combination with α3 and possibly other subunits.19–21 Furthermore, αBgt-sensitive α7 nAChRs are expressed by rabbit cone bipolar, amacrine, and ganglion cells,22 suggesting that activation of α7 nAChRs by ACh may affect information processing in multiple retinal circuits. Consistent with inner retinal localization of nAChRs, physiological studies demonstrate that the frog ERG b-wave is inhibited by ACh,23 possibly through a cholinergic-glycinergic feedback loop24,25; the cat b-wave is first enhanced and then rapidly inhibited by ACh,26 suggesting the activation of multiple upstream nAChR subtypes. In rhesus monkey, the pattern ERG is enhanced by the ACh precursor L-α-glyceryl-phosphorylcholine.27 Although cholinergic mechanisms mediated by nAChRs also influence experimental retinal neovascularization28 and are involved in refractive development,29–31 a major gap in understanding the role of ACh in the normal and diseased retina is the limited information concerning the expression of nAChRs in non-human primate and human retinas. We report the results of RT-PCR and immunohistochemical studies of nAChR subunit expression in Rhesus monkey retina and discuss these findings in comparison to previous observations in the retinas of other mammals.

Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

Abstract: High-affinity, beta2-subunit-containing (beta2) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2 nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2 nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2 nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

Abstract: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol˙mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits.

Association of sequence variations in the gene encoding insulin-like growth factor binding protein 5 with adiponectin

Abstract: Insulin-like growth factor binding protein 5 (IGFBP5) binds to IGF and thus modulates IGF signaling pathway. We have shown earlier that the IGFBP5 gene was downregulated in the adipose tissue after 12-week carbohydrate diet with low insulinemic response. The aim was to examine the putative contribution of genetic variation of the IGFBP5 gene to the characteristics of metabolic syndrome and incidence of type 2 diabetes (T2DM) in the Finnish Diabetes Prevention Study (DPS). DPS is a longitudinal study where 522 subjects with impaired glucose tolerance were randomized to either lifestyle intervention group or control group. DNA was available from 507 subjects (mean body mass index (BMI) 31.2±4.5 kg/m2, age 55±7 years). The eight single-nucleotide polymorphisms (SNPs) were selected from HapMap database and genotyped by Taqman allelic discrimination protocol. The main results were confirmed in a larger cross-sectional study population (METSIM). In addition, the gene expression of IGFBP5 was studied in two previously published study populations (FUNGENUT and GENOBIN) of 124 subjects with insulin resistance (BMI 32.2±3.5 kg/m2, age 57.7±7.4 years). Three out of eight IGFBP5 markers (rs9341234, rs3276 and rs11575134) were significantly associated with circulating adiponectin concentrations in men. Furthermore, mRNA expression studies of subcutaneous adipose tissue showed that mRNA concentrations of IGFBP5 correlated with adiponectin concentrations in all subjects and in women. None of the IGFBP5 SNPs were associated with T2DM. Our findings show that IGFBP5 has a gender-specific association with adiponectin, which may modulate the development of metabolic syndrome.

Mutations of Cytosolic Loop Residues Impair Assembly and Maturation of α7 Nicotinic Acetylcholine Receptors

Abstract: Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein resistant to inhibitors of cholineesterase-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH-SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes.