New tools for targeted disruption of cholinergic synaptic transmission in Drosophila melanogaster.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson's disease, Alzheimer's disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR's ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling

Inhibition of cholinergic pathways in Drosophila melanogaster by α-conotoxins

Nicotinic acetylcholine receptors (nAChRs) play a pivotal role in synaptic transmission of neuronal signaling pathways and are fundamentally involved in neuronal disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia. In vertebrates, cholinergic pathways can be selectively inhibited by α-conotoxins; we show that in the model organism Drosophila, the cholinergic component of the giant fiber system is inhibited by α-conotoxins MII, AuIB, BuIA, EI, PeIA, and ImI. The injection of 45 pmol/fly of each toxin dramatically decreases the response of the giant fiber to dorsal longitudinal muscle (GF-DLM) connection to 20 ± 13.9% for MII; 26 ± 13.7% for AuIB, 12 ± 9.9% for BuIA, 30 ± 11.3% for EI, 1 ± 1% for PeIA, and 34 ± 15.4% for ImI. Through bioassay-guided fractionation of the venom of Conus brunneus, we found BruIB, an α-conotoxin that inhibits Drosophila nicotinic receptors but not its vertebrate counterparts. GF-DLM responses decreased to 43.7 ± 8.02% on injection of 45 pmol/fly of BruIB. We manipulated the Dα7 nAChR to mimic the selectivity of its vertebrate counterpart by placing structurally guided point mutations in the conotoxin-binding site. This manipulation rendered vertebrate-like behavior in the Drosophila system, enhancing the suitability of Drosophila as an in vivo tool to carry out studies related to human neuronal diseases

Refractory period and following frequency Dα7 nAChR subunits in wild type background.

<p>(A) Scatterplot of Refractory Periods (Twin pulses) of DLM recordings, when the GF was stimulated in the brain. Genotypes shown are WT-Dα7, Dα7-D77T/L117Q/I196P, Dα7-D197A and the Dα7-Y195T expressed in wild type background as well as A307 wildtype control. (B) Scatterplot of Following frequency of DLM recordings, when the GF was stimulated in the brain. Genotypes shown are WT-Dα7, Dα7-D77T/L117Q/I196P, Dα7-D197A and the Dα7-Y195T expressed in wild type background as well as A307 wildtype control. *p<0.05, n = 24 DLMs for all treatments and genotypes.</p

Refractory period and following frequency of Dα7 nAChR subunits in Dα7 null mutant background.

<p>(A) Scatterplot of Refractory Periods (Twin pulses) of DLM recordings, when the GF was stimulated in the brain. Genotypes shown are WT-Dα7, Dα7-D77T/L117Q/I196P, Dα7-D197A and the Dα7-Y195T expressed in a Dα7 null background. Wildtype control (A307), negative control gfA^PΔEY6/>(n = 30 DLMs) and gfA^PΔEY6/+ flies are shown as well. (B) Scatterplot of maximum Following Frequency of DLM recordings, when the GF was stimulated in the brain. Genotypes are Dα7, Dα7-D77T/L117Q/I196P, Dα7-D197A and the Dα7-Y195T expressed in a Dα7 null background. Wildtype control (A307), negative control gfA^PΔEY6/>(n = 20 DLMs) and gfA^PΔEY6/+ flies are shown as well. *p<0.05, n = 24 DLMs for all treatments and genotypes unless otherwise noted.</p

Sequence alignment of ligand-binding domain of Dα7 with Human α7 nAChR subunit.

<p>There is a 48% amino acid identity of the entire subunit sequences. Within the ligand-binding domain, there is an 81% amino acid homology, with a 58% amino acid identity. Key residues for agonist binding are highlighted in yellow and mutated residues are in red. An * (asterisk) below residue alignment denotes positions that have a identical residues. A colon denotes groups with strongly similar properties and a scoring of >0.5 in the Gonnet PAM 250 matrix. A period denotes groups with weakly similar properties and a scoring of ≤0.5 in the Gonnet PAM 250 matrix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064685#pone.0064685-Pettersen1" target="_blank">[28]</a>.</p

Structural models of mutant Dα7 nAChR ligand-binding domain.

<p>(A) Triple mutant D77T/L117Q/I196P: key agonist binding residues (yellow) and point mutations (red) are highlighted. (B) D197A mutant: key agonist binding residues (yellow) and single point mutation (red) are highlighted. (C) Y195T mutant: single point mutation (light blue) overlapping one key agonist binding residue and other key agonist binding residues (yellow) are highlighted.</p

Noribogaine is a G-protein biased κ-opioid receptor agonist

Noribogaine is the long-lived human metabolite of the anti-addictive substance ibogaine. Noribogaine efficaciously reaches the brain with concentrations up to 20 μM after acute therapeutic dose of 40 mg/kg ibogaine in animals. Noribogaine displays atypical opioid-like components in vivo, anti-addictive effects and potent modulatory properties of the tolerance to opiates for which the mode of action remained uncharacterized thus far. Our binding experiments and computational simulations indicate that noribogaine may bind to the orthosteric morphinan binding site of the opioid receptors. Functional activities of noribogaine at G-protein and non G-protein pathways of the mu and kappa opioid receptors were characterized. Noribogaine was a weak mu antagonist with a functional inhibition constants (Ke) of 20 μM at the G-protein and β-arrestin signaling pathways. Conversely, noribogaine was a G-protein biased kappa agonist 75% as efficacious as dynorphin A at stimulating GDP-GTP exchange (EC50 = 9 μM) but only 12% as efficacious at recruiting β-arrestin, which could contribute to the lack of dysphoric effects of noribogaine. In turn, noribogaine functionally inhibited dynorphin-induced kappa β-arrestin recruitment and was more potent than its G-protein agonistic activity with an IC50 of 1 μM. This biased agonist/antagonist pharmacology is unique to noribogaine in comparison to various other ligands including ibogaine, 18-MC, nalmefene, and 6′-GNTI. We predict noribogaine to promote certain analgesic effects as well as anti-addictive effects at effective concentrations >1 μM in the brain. Because elevated levels of dynorphins are commonly observed and correlated with anxiety, dysphoric effects, and decreased dopaminergic tone, a therapeutically relevant functional inhibition bias to endogenously released dynorphins by noribogaine might be worthy of consideration for treating anxiety and substance related disorders. •The anti-addictive drug noribogaine was functionally characterized at the opioid receptors.•Noribogaine is a G-protein biased kappa opioid agonist.•Noribogaine is also a mu antagonist and a beta-arrestin biased kappa antagonist.•The unique pharmacological profile of noribogaine may explain its atypical effects on the opioid system in vivo

Transforming conotoxins into cyclotides: Backbone cyclization of P-superfamily conotoxins

Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs