Inhibiting cellular uptake of mutant huntingtin using a monoclonal antibody: Implications for the treatment of Huntington's disease.

Huntington's disease (HD) is caused by a highly polymorphic CAG trinucleotide expansion in the gene encoding for the huntingtin protein (HTT). The resulting mutant huntingtin protein (mutHTT) is ubiquitously expressed but also exhibits the ability to propagate from cell-to-cell to disseminate pathology; a property which may serve as a new therapeutic focus. Accordingly, we set out to develop a monoclonal antibody (mAB) targeting a particularly exposed region close to the aa586 caspase-6 cleavage site of the HTT protein. This monoclonal antibody, designated C6-17, effectively binds mutHTT and is able to deplete the protein from cell culture supernatants. Using cell-based assays, we demonstrate that extracellular secretion of mutHTT into cell culture media and its subsequent uptake in recipient HeLa cells can be almost entirely blocked by mAB C6-17. Immunohistochemical stainings of post-mortem HD brain tissue confirmed the specificity of mAB C6-17 to human mutHTT aggregates. These findings demonstrate that mAB C6-17 not only successfully engages with its target, mutHTT, but also inhibits cell uptake suggesting that this antibody could interfere with the pathological processes of mutHTT spreading in vivo.

Pharmacochaperoning of the A1 adenosine receptor is contingent on the endoplasmic reticulum.

Exchanging each of the conserved aromatic residues of the NPxxY(x)(5,6)F sequence (at the boundary of helices 7 and 8) generated variants of the A(1) adenosine receptor that were retained within the cell. The mutations disconnected a link between alpha-helix 7 and cytosolic helix 8, likely destabilizing the structure of the proximal carboxyl terminus. The mutant receptors were rescued by incubation of cells with a pharmacochaperone, a membrane-permeable ligand that homosterically binds to the receptor; pharmacochaperoning restored the density of functional receptors at the plasma membrane. The following observations support the assumption that retention and the site of pharmacochaperone action were within bounds of the endoplasmic reticulum (ER): 1) the retained receptor colocalized with an ER marker; 2) pharmacochaperoning initiated receptor transfer to Golgi stacks; and 3) the inhibitor of glycoprotein synthesis tunicamycin suppressed receptor chaperoning. Our data are consistent with the hypothesis that pharmacochaperoning stabilizes the structure of late folding intermediates and lifts a block on maturation, allowing the receptors to exit from the ER. We suggest that the ER-associated 40-kDa heat shock protein family member D(1) receptor interacting protein 78 (DRiP78; M(r), approximately 78,000) represents a model executor of quality control. Overexpressed DRiP78 interacted physically with the A(1) receptor, inhibited export to the plasma membrane, and in this action was selective for the mutants relative to the wild-type receptor. Both agonist and antagonist were effective chaperone ligands. Thus, occupancy of the binding pocket corrected the mutation-induced disorder, indicating a mutual impingement of the transmembrane domain and the proximal carboxyl terminus in establishing the stable receptor fold.

Effects of duramycin on cardiac voltage-gated ion channels.

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 microM did not generate any effects on these currents. Concentrations >or=0.3 microM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (>or=3.3 microM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias.

Quantitative in vitro and in vivo models to assess human IgE B cell receptor crosslinking by IgE and EMPD IgE targeting antibodies.

Targeting plasma IgE by therapeutic mABs like Omalizumab (Xolair®) is current clinical practice for severe allergic conditions or other IgE related diseases like chronic urticaria. As an alternative to soluble IgE targeting, IgE supply can be lowered by targeting the Extracellular Membrane Proximal Domain (EMPD) of the IgE B cell receptor (BCR) present on IgE switched B cells. This ultimately leads to apoptosis of these cells upon IgE BCR crosslinking. Since tools to selectively assess the efficacy of IgE BCR crosslinking by IgE targeting antibodies are limited, a readily quantifiable cell model was developed that allows to specifically address IgE BCR crosslinking activity in vitro. The new cell model allowed for a direct quantitative comparison of anti-EMPD IgE therapeutic prototype antibody 47H4 with anti-IgE(Ce3) directed therapeutic antibody Omalizumab and with a newly selected anti-human EMPD IgE monoclonal antibody, designated mAB 15cl12. Furthermore, a complementing mouse model was developed that allows for in vivo validation of antibodies addressing human EMPD IgE. It carries a targetable humanized EMPD IgE sequence that has been introduced by seamless genomic replacement of the endogenous EMPD encoding sequence. The model allowed to directly compare IgE lowering activity of two anti-human EMPD IgE therapeutic antibodies in vivo. Our tools provide the means for quantitative assessment of IgE BCR crosslinking activity which is increasingly gaining attention with respect to forthcoming second generation anti-IgE clinical candidates such as Ligelizumab or other clinical candidates featuring additional effector functions such as IgE BCR crosslinking activity.

The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism.

Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling.

The carboxyl terminus (C-tail) of G protein-coupled receptors is divergent in length and structure and may represent an individualized cytoplasmic domain. By progressively truncating the A1 adenosine receptor, a Gi/o-coupled receptor with short cytoplasmic stretches, we identify two inherent functions of the C-tail, namely a role in receptor export from the endoplasmic reticulum (ER) and a role in G protein coupling. Deletion of the last 22 and 26 amino acids (of 36) reduced and completely abolished surface expression of the receptor, respectively. The severely truncated receptors were retained in the ER and failed to bind ligands. If overexpressed, even a substantial portion of the full-length receptor was retained in the ER in a form that was not functional. These data indicate that folding is rate limiting in export from the ER and that the proximal segment of the carboxyl terminus provides a docking site for the machinery involved in folding and quality control. In addition, the proximal portion is also important in G protein coupling. This latter role was unmasked when the distal portion of the C-tail (the extreme 18 amino acids, including a palmitoylated cysteine) had been removed; the resulting receptor was functional and transferred the agonist-mediated signal more efficiently than the full-length receptor. Signaling was enhanced because the coupling affinity increased (by 3-fold), which translated into a higher agonist potency. Thus, the distal portion of the carboxyl terminus provides for an autoinhibitory restraint, presumably by folding back and preventing G protein access to the proximal part of the C-tail.

Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y receptor-mediated inhibition of voltage-gated Ca2+ channels.

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.