Sara Fuchs

D3 dopamine receptor-deficient mouse: evidence for reduced anxiety.

Mice without functional D3 dopamine receptors were examined in two animal models for anxiety: the open-field test and the elevated plus-maze test. In the open field, D3 receptor-deficient mice (D3-/-) entered the center significantly more often than normal (D3+/+) littermates, suggesting an anxiolytic-like effect of the D3 receptor mutation. Consistent with this finding, D3-/- mice entered open arms of the plus maze significantly more often and longer than D3+/+ littermates, but did not differ in closed-arm entries, an index of general activity. Heterozygous (D3 +/-) animals showed intermediate behavioral changes. We interpret these results as indicative of reduced anxiety in mice without D3 receptors. Our findings thus suggest that D3 dopamine receptors are involved in the regulation of anxiety.

In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.

βγ Dimers Mediate Synergy of Dopamine D2 and Adenosine A2 Receptor-Stimulated PKA Signaling and Regulate Ethanol Consumption

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. We report that dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester betagamma dimers prevent synergy. Importantly, overexpression of a betagamma inhibitor peptide in the nucleus accumbens strikingly reduces sustained alcohol consumption. We propose that synergy of D2 and A2 confers ethanol hypersensitivity and that betagamma dimers are required for voluntary drinking.

Immunochemical studies on acetylcholine receptor from Torpedo californica.

Abstract Acetylcholine receptor (AChR) was purified from electric organ tissue of Torpedo california by solubilization of membrane fragments with 1% Triton X-100 followed by affinity chromatography on a Naja naja siamensis neurotoxin-Sepharose resi. The molecular and pharmacological properties of the purified recerptor were determined, and it was shown to have a specific activity of 8–12 nmoles toxin bound per mg protein, corresponding to 80,000–125,000 daltons per toxin-binding site. Experimental autoimmune myasthenia gravis was consistently induced in rabbits following a single injection of purified AChR. The cellular and humoral immune responses to the purifed AChR were analyzed in the injected animals. By lymphocyte transformation in vitro it was shown that such animals responded not only to the homologous immunogen— Torpedo AChR—but also to extracts of rabbit skeletal muscle or thymus. Humoral rabbit anti-AChR antibodies gave one precipitation line with AChR in immunodiffusion and immunoelectrophoresis. The antigenic determinants against which antibodies were produced are different from the toxin-binding sites of the AChR. However, anti-AChR antibodies inhibit toxin-binding, probably due to steric hindrance. A possible molecular model is presented for explaining the relationship between the physiological active site, the ‘myasthenic determinant’ and other antigenic determinants on the AChR molecule.

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

Genetics of Immunoresponsiveness to Natural Antigens in the Mouse

The nicotinic acetylcholine receptor (AChR) has been extensively studied both from the structural and biologic points of view. It is the only receptor for a neurotransmitter that has been purified and can be prepared in substantial amounts. Characterization of this component has been greatly advanced by the availability of AChR in large quantities in electric organs of electric fish (Nachmansohn, 1959) and by the use of a-neurotoxins from elapid snake venoms which bind specifically and with a high affinity to the nicotinic acetylcholine receptor (Lee, 1972).

HUMORAL ANTIBODIES TO ACETYLCHOLINE RECEPTOR IN PATIENTS WITH MYASTHENIA GRAVIS

Sera from patients with myasthenia gravis (M.G.) were studied by the quantitative micro-scale complement-fixation assay for the presence of humoral antibodies against acetylcholine receptor (AChR). The purified receptor was extracted from the electrogenic tissue of the electric ray, Torpedo californica. A significant difference in the antibody titres was observed between myasthenic and non-myasthenic patients. Out of fifteen patients with myasthenia gravis, at least 12 (80%) had antibodies against AChR. Only one case out of twenty-four controls had an indication of anti-receptor antibodies. In view of observations on the role of AChR as the autoantigen in myasthenia gravis, such antibodies may have significance in producing the neuro-muscular block characteristic of the disease.

Dopamine Receptors and Dopamine Transporter in Brain Function and Addictive Behaviors: Insights from Targeted Mouse Mutants

Recent advances in molecular biology have resulted in a number of genetically manipulated mice with defined changes at dopamine receptor and the dopamine transporter (DAT) loci. Mice with targeted mutations at the D1 receptor (D1R) are growth-retarded and show downregulated expression of dynorphin and substance P. Behavioral assessment indicates that mutants have deficiencies in spatial learning and initiating movement, as well as in responding to novel stimuli. D1R mutants do not become locomotor activated with cocaine or show upregulated immediate early gene (IEG) expression, but D2 receptor-dependent IEG changes are intact. Acute cocaine administration increases substance P levels, suggesting that striatal expression of this neuropeptide can be modulated by D1R-independent processes. Failure of locomotor activation is also seen with repeated amphetamine treatment. Surprisingly, D1R-deficient mice retain cocaine-conditioned place preference. In contrast, D2 receptor knockout mice are bradykinetic, show increased striatal enkephalin expression and an absence of opiate rewarding effects. D3 receptor mutants are hyperactive when assessed in an exploratory assay and display reduced anxiety-associated behavior in an elevated plus maze test. The recently described D4 receptor homozygous mutants exhibit a reduction in baseline locomotor activity and were shown to be supersensitive to the locomotor activating effects of alcohol and psychostimulant drugs. As expected, DAT knockout mice are hyperactive and do not respond to cocaine or amphetamine. The observation that D2 and D4 dopamine receptor and DAT mutants show compensatory effects, together with the complicating issue of their hybrid genetic background may temper conclusions regarding the direct effects of the targeted mutation on phenotype.

Adenylyl Cyclase Interaction with the D2 Dopamine Receptor Family; Differential Coupling to Gi, Gz, and Gs

Abstract1.The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity.2.COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the α subunit of Gz. PTX treatment was employed to inactivate endogenous αi, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells.3.D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively.4.We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX.5.Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.

Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent Ab-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1-type cells and costimulatory factors such as CD40 ligand (CD40L) contribute to disease pathogenesis by producing proinflammatory cytokines and by activating autoreactive B cells. In this study we demonstrate the capacity of CD40L blockade to modulate EAMG, and analyze the mechanism underlying this disease suppression. Anti-CD40L Abs given to rats at the chronic stage of EAMG suppress the clinical progression of the autoimmune process and lead to a decrease in the AChR-specific humoral response and delayed-type hypersensitivity. The cytokine profile of treated rats suggests that the underlying mechanism involves down-regulation of AChR-specific Th1-regulated responses with no significant effect on Th2- and Th3-regulated AChR-specific responses. EAMG suppression is also accompanied by a significant up-regulation of CTLA-4, whereas a series of costimulatory factors remain unchanged. Adoptive transfer of splenocytes from anti-CD40L-treated rats does not protect recipient rats against subsequently induced EAMG. Thus it seems that the suppressed progression of chronic EAMG by anti-CD40L treatment does not induce a switch from Th1 to Th2/Th3 regulation of the AChR-specific immune response and does not induce generation of regulatory cells. The ability of anti-CD40L treatment to suppress ongoing chronic EAMG suggests that blockade of CD40L may serve as a potential approach for the immunotherapy of MG and other Ab-mediated autoimmune diseases.