MacDonald J. Christie

Dendritic Function of Tau Mediates Amyloid-β Toxicity in Alzheimer's Disease Mouse Models

Alzheimers disease (AD) is characterized by amyloid-beta (Abeta) and tau deposition in brain. It has emerged that Abeta toxicity is tau dependent, although mechanistically this link remains unclear. Here, we show that tau, known as axonal protein, has a dendritic function in postsynaptic targeting of the Src kinase Fyn, a substrate of which is the NMDA receptor (NR). Missorting of tau in transgenic mice expressing truncated tau (Deltatau) and absence of tau in tau(-/-) mice both disrupt postsynaptic targeting of Fyn. This uncouples NR-mediated excitotoxicity and hence mitigates Abeta toxicity. Deltatau expression and tau deficiency prevent memory deficits and improve survival in Abeta-forming APP23 mice, a model of AD. These deficits are also fully rescued with a peptide that uncouples the Fyn-mediated interaction of NR and PSD-95 in vivo. Our findings suggest that this dendritic role of tau confers Abeta toxicity at the postsynapse with direct implications for pathogenesis and treatment of AD.

How opioids inhibit GABA-mediated neurotransmission

The midbrain region periaqueductal grey (PAG) is rich in opioid receptors and endogenous opioids and is a major target of analgesic action in the central nervous system. It has been proposed that the analgesic effect of opioids on the PAG works by suppressing the inhibitory influence of the neurotransmitter GABA (γ-aminobutyric acid) on neurons that form part of a descending antinociceptive pathway. Opioids inhibit GABA-mediated (GABAergic) synaptic transmission in the PAG and other brain regions by reducing the probability of presynaptic neurotransmitter release,, but the mechanisms involved remain uncertain. Here we report that opioid inhibition of GABAergic synaptic currents in the PAG is controlled by a presynaptic voltage-dependent potassium conductance. Opioid receptors of the μ type in GABAergic presynaptic terminals are specifically coupled to this potassium conductance by a pathway involving phospholipase A2, arachidonic acid and 12-lipoxygenase. Furthermore, opioid inhibition of GABAergic synaptic transmission is potentiated by inhibitors of the enzymes cyclooxygenase and 5-lipoxygenase, presumably because more arachidonic acid is available for conversion to 12-lipoxygenase products. These mechanisms account for the analgesic action of cyclooxygenase inhibitors in the PAG and their synergism with opioids.

Regulation of μ-opioid receptors: Desensitization, phosphorylation, internalization, and tolerance

Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance.

Cellular neuroadaptations to chronic opioids: tolerance, withdrawal and addiction

A large range of neuroadaptations develop in response to chronic opioid exposure and these are thought to be more or less critical for expression of the major features of opioid addiction: tolerance, withdrawal and processes that may contribute to compulsive use and relapse. This review considers these adaptations at different levels of organization in the nervous system including tolerance at the μ‐opioid receptor itself, cellular tolerance and withdrawal in opioid‐sensitive neurons, systems tolerance and withdrawal in opioid‐sensitive nerve networks, as well as synaptic plasticity in opioid sensitive nerve networks. Receptor tolerance appears to involve enhancement of mechanisms of receptor regulation, including desensitization and internalization. Adaptations causing cellular tolerance are more complex but several important processes have been identified including upregulation of cAMP/PKA and cAMP response element‐binding signalling and perhaps the mitogen activated PK cascades in opioid sensitive neurons that might not only influence tolerance and withdrawal but also synaptic plasticity during cycles of intoxication and withdrawal. The potential complexity of network, or systems adaptations that interact with opioid‐sensitive neurons is great but some candidate neuropeptide systems that interact with μ‐opioid sensitive neurons may play a role in tolerance and withdrawal, as might activation of glial signalling. Implication of synaptic forms of learning such as long term potentiation and long term depression in opioid addiction is still in its infancy but this ultimately has the potential to identify specific synapses that contribute to compulsive use and relapse.

Conus Venom Peptide Pharmacology

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (∼0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones.

The actions of the endogenous ORL1‐receptor (opioid receptor‐like1) ligand, nociceptin, on the membrane properties of rat dorsal raphe nucleus neurones were examined by use of whole‐cell patch clamp recording in brain slices. Nociceptin produced an outward current in all neurones tested, with an EC50 of 12 ± 2 nM. Dynorphin A (100 nM to 1 μm) produced little outward current. Outward currents reversed polarity near the predicted K+ equilibrium potential in both 2.5 mM (measured/predicted = −105 mV/ −104 mV) and 6.5 mM (measured/predicted = −80 mV/ −77 mV) extracellular K+. The conductance increase was larger between −120 and −130 mV than between −70 and −80 mV, demonstrating that the nociceptin‐induced K current was due to an increased inwardly rectifying K conductance. The outward current produced by nociceptin was similar to, and occluded by, high concentrations of baclofen, demonstrating actions on the same population of K channels. Naloxone (1 μm) failed to inhibit outward currents produced by nociceptin. These results are consistent with the reported high density of ORL1 receptor mRNA in dorsal raphe nucleus and with inhibitory actions of nociceptin in cells expressing ORL1.

Heteropolymeric potassium channels expressed in xenopus oocytes from cloned subunits

Voltage-dependent potassium currents were measured in Xenopus oocytes previously injected with RNAs generated in vitro from each of three cloned cDNAs (RBK1, RBK2, and RGK5). The currents differed in their sensitivities to blockade by tetraethylammonium (TEA; respective KDs 0.3, greater than 100, and 10 mM) and in their inactivation during a depolarizing pulse. Injections of RNA combinations (RBK1/RBK2 and RBK1/RGK5) caused currents that had TEA sensitivities different from those expected from the sum, in any proportion, of the two native channels. It is concluded that novel potassium channels are formed by the oocytes injected with two RNAs, presumably by heteropolymerization of subunits; such heteropolymerization would contribute functional diversity to voltage-dependent potassium channels in addition to that provided by a large gene family.

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1 In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid‐like receptor ORL1, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2 When locus coeruleus neurones were voltage clamped to −60 mV, application of nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 μm) or thiorphan (2 μm). 3 The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at −123 mV, more negative than the predicted K+ reversal potential of −105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4 Application of a high concentration of nociceptin (3 μm) occluded the current produced by simultaneous application of high concentrations of Met‐enkephalin (10 μm), (3 μm) somatostatin and UK 14304 (3 μm), indicating that nociceptin activated the same conductance as μ‐opioid and somatostatin receptors and α2‐adrenoceptors. 5 The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb′s estimated from 2 cells of −4.23 and −4.33. The μ‐opioid antagonist, CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Pen‐Thr‐NH2, 1 μm), the opioid antagonist, nalorphine (30 μm), or the somatostatin antagonist, CPP (cyclo(7‐aminoheptanoyl‐Phe‐D‐Tip‐Lys‐Thr[Bzl]) 3 μm) did not affect the nociceptin‐induced current. 6 Dynorphin A (3 μm), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM‐1 μm). 7 Continuous application of nociceptin (3 μm) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met‐enkephalin (30 μm) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met‐enkephalin in the same cells. Conversely, continuous application of Met‐enkephalin (30 μm) resulted in a decrease of Met‐enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 μm) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8 Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.

Presynaptic inhibitory action of opioids on synaptic transmission in the rat periaqueductal grey in vitro.

1. The actions of opioids on synaptic transmission in rat periaqueductal grey (PAG) neurones were examined using whole‐cell patch‐clamp recordings in brain slices. 2. Methionine enkephalin (ME; 10 microM) inhibited evoked GABAergic inhibitory postsynaptic currents (IPSCs) by 57%, non‐NMDA excitatory postsynaptic currents (EPSCs) by 60%, and NMDA EPSCs by 43% in PAG neurones. This inhibition was associated with an increase in paired‐pulse facilitation, was mimicked by the mu‐agonist DAMGO (1‐3 microM) and abolished by naloxone (1 microM). Neither the kappa‐agonist U69593 (1‐3 microM), nor the delta‐agonist DPDPE (3‐10 microM) had any specific actions on evoked PSCs. 3. ME decreased the frequency of spontaneous miniature, action potential‐independent postsynaptic currents (mIPSCs by 65%, mEPSCs by 54%) in all PAG neurones, but had no effect on their amplitude distributions. The reduction in mIPSC frequency persisted in nominally Ca(2+)‐free, high‐Mg2+ (10 mM) solutions, which also contained Cd2+ (100 microM), or Ba2+ (10 mM). Opioid inhibition of mIPSC frequency is unlikely to be mediated by presynaptic Ca2+ or K+ conductances which are sensitive to extracellular Cd2+ or Ba2+. 4. In a subpopulation of PAG neurones, ME increased a Ba(2+)‐sensitive K+ conductance at potentials below ‐97 mV. Opioids inhibited both GABAergic and glutamatergic synaptic transmission in all PAG neurones, independent of any postsynaptic opioid sensitivity. 5. These observations are consistent with, but only partially support, the opioid disinhibition model of PAG‐induced analgesia. mu‐Opioids also have the potential to modulate the behavioural and autonomic functions of the PAG via modulation of both inhibitory and excitatory presynaptic mechanisms, as well as postsynaptic mechanisms.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.