Recombinant glycine receptors: stoichiometry and kinetics

Glycine receptors (GlyR) are anion-permeable channels that belong to the pentameric ligand-gated ion channel family. Different GlyR subtypes are known. The main synaptic form is thought to be α1β heteropentamers which mediate fast synaptic inhibition in the adult spinal cord and brainstem. Data on recombinant receptors suggest two possible stoichiometries for this subtype, 2α1:3β and 3α1:2β. Evidence for the first comes from experiments on oocytes, whereas a study in mammalian cells favours the latter, raising the possibility that stoichiometry depends on the expression system. Here, we assess the stoichiometry of α1β GlyRs in Xenopus oocytes using two different electrophysiological approaches. The first involves the use of a reporter mutation at the conserved 9΄ position of the pore-lining domain. In other receptors, this mutation shifts agonist sensitivity in proportion to the number of mutated subunits. Recordings from mutant receptors failed to point towards one or the other stoichiometry. The second approach involved single-channel recordings from conductance mutants. This approach was also inconclusive for stoichiometry. However, we provide evidence that oocytes are not a suitable expression system for the study of heteromeric glycine receptors as they are highly prone to contamination by homomers. α2 homomeric GlyRs are predominant early in development and are replaced by α1 subunits in the first postnatal days. We investigated the activation mechanism of these channels in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α2 channels can open only when all binding sites are occupied by glycine, and only after the channel undergoes a conformational change ('flip') that links binding to gating. Macroscopic data favour a two binding site model. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments when desensitization is included in the model

Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease

Alterations of the dopaminergic (DAergic) system are frequently reported in Alzheimer’s disease (AD) patients and are commonly linked to cognitive and non-cognitive symptoms. However, the cause of DAergic system dysfunction in AD remains to be elucidated. We investigated alterations of the midbrain DAergic system in the Tg2576 mouse model of AD, overexpressing a mutated human amyloid precursor protein (APPswe). Here, we found an age-dependent DAergic neuron loss in the ventral tegmental area (VTA) at pre-plaque stages, although substantia nigra pars compacta (SNpc) DAergic neurons were intact. The selective VTA DAergic neuron degeneration results in lower DA outflow in the hippocampus and nucleus accumbens (NAc) shell. The progression of DAergic cell death correlates with impairments in CA1 synaptic plasticity, memory performance and food reward processing. We conclude that in this mouse model of AD, degeneration of VTA DAergic neurons at pre-plaque stages contributes to memory deficits and dysfunction of reward processing

DMSO and Temperature Contributions to Synthesis of Silver Nano-Particles by the Bacterium Shewanella oneidensis

Nanomaterial are widely used in different areas such as optical device, drug delivery, chemicals, mechanics, magnetics, catalysis, energy science, Nano therapeutics and space industries depend on the special physical properties. However, most methods to produce nanoparticles are expensive or environmental unfriendly which can involve in toxic chemical. Another reason is that the nanoparticles from bio-based protocols are hydrophilic which is compatible with biological materials. In this project, we chose Shewanella oneidensis which is Gram-negative bacterium as the organism to produce sliver nanoparticles from sliver nitrate solution. The mechanism of bacterial of ion metal ion reduction to stable metal nanoparticles is unclear, but the NADH-dependent reeducates, quinines, and soluble electron-shuttles are thought to play an important role in metal reduction. This research focused on the temperature and DMSO affects the synthesis of silver nanoparticles by Shewanella Oneidensis. At various temperatures, the bio-activity of bacterium is different which can affect the silver nanoparticles reducing rate and the spherical size and nanoparticle geometry. DMSO is an aprotic, polar solvent which can penetrate skin and other membranes without damaging the cells. Due to this property of DMSO, DMSO was utilized as a co-solvent, which may change biosynthesis of silver nanoparticles. The synthesis processes were carried out at different temperatures and DMSO concentration and the nanoparticle formation monitored by using UV-vis spectrometer scans of the aqueous layer of reaction at 0 hr, 24 hr and 48 hr.https://ecommons.udayton.edu/stander_posters/1429/thumbnail.jp

Human alpha 3 beta 4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

Background: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the alpha 3 and beta 4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the alpha 5 and/or beta 2 subunits. We compared the properties of human alpha 3 beta 4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and alpha:beta subunit ratio.Methodology/Principal Findings: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the alpha subunit, as reported for alpha 4 beta 2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike alpha 4 beta 2 channels, for alpha 3 beta 4 receptors the putative two-alpha form is the predominant one in oocytes (at 1:1 alpha:beta cRNA ratio). This two-alpha form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-alpha form is the predominant one in HEK cells transfected with a 1:1 alpha:beta DNA ratio or in oocytes at 9:1 alpha:beta RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-alpha form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-alpha form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/Significance: Like other neuronal nicotinic receptors, the alpha 3 beta 4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected

Dysfunctional dopaminergic neurotransmission in asocial BTBR mice

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by pronounced social and communication deficits and stereotyped behaviours. Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients. However, the neurobiological and molecular determinants of these deficits remain undetermined. Mouse models recapitulating ASD-like phenotypes could help generate hypotheses about the origin and neurophysiological underpinnings of clinically relevant traits. Here we used functional magnetic resonance imaging (fMRI), behavioural and molecular readouts to probe dopamine neurotransmission responsivity in BTBR T+ Itpr3tf/J mice (BTBR), an inbred mouse line widely used to model ASD-like symptoms owing to its robust social and communication deficits, and high level of repetitive stereotyped behaviours. C57BL/6J (B6) mice were used as normosocial reference comparators. DA reuptake inhibition with GBR 12909 produced significant striatal DA release in both strains, but failed to elicit fMRI activation in widespread forebrain areas of BTBR mice, including mesolimbic reward and striatal terminals. In addition, BTBR mice exhibited no appreciable motor responses to GBR 12909. DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals. Overall these results document profoundly compromised DA D2-mediated neurotransmission in BTBR mice, a finding that is likely to have a role in the distinctive social and behavioural deficits exhibited by these mice. Our results call for a deeper investigation of the role of dopaminergic dysfunction in mouse lines exhibiting ASD-like phenotypes, and possibly in ASD patient populations

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50=12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity

Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease

Alterations of the dopaminergic (DAergic) system are frequently reported in Alzheimer's disease (AD) patients and are commonly linked to cognitive and non-cognitive symptoms. However, the cause of DAergic system dysfunction in AD remains to be elucidated. We investigated alterations of the midbrain DAergic system in the Tg2576 mouse model of AD, overexpressing a mutated human amyloid precursor protein (APPswe). Here, we found an age-dependent DAergic neuron loss in the ventral tegmental area (VTA) at pre-plaque stages, although substantia nigra pars compacta (SNpc) DAergic neurons were intact. The selective VTA DAergic neuron degeneration results in lower DA outflow in the hippocampus and nucleus accumbens (NAc) shell. The progression of DAergic cell death correlates with impairments in CA1 synaptic plasticity, memory performance and food reward processing. We conclude that in this mouse model of AD, degeneration of VTA DAergic neurons at pre-plaque stages contributes to memory deficits and dysfunction of reward processing

Passive immunotherapy for N-truncated tau ameliorates the cognitive deficits in two mouse AD models

Clinical and neuropathological studies have shown that tau pathology better correlates with the severity of dementia than amyloid plaque burden, making tau an attractive target for the cure of Alzheimer\u2019s disease. We have explored whether passive immunization with the 12A12 monoclonal antibody (26\u201336aa of tau protein) could improve the Alzheimer\u2019s disease phenotype of two well-established mouse models, Tg2576 and 3xTg mice. 12A12 is a cleavage-specific monoclonal antibody which selectively binds the pathologically relevant neurotoxic NH226-230 fragment (i.e. NH2htau) of tau protein without cross-reacting with its full-length physiological form(s). We found out that intravenous administration of 12A12 monoclonal antibody into symptomatic (6 months old) animals: (i) reaches the hippocampus in its biologically active (antigen-binding competent) form and successfully neutralizes its target; (ii) reduces both pathological tau and amyloid precursor protein/amyloid\u3b2 metabolisms involved in early disease-associated synaptic deterioration; (iii) improves episodic-like type of learning/memory skills in hippocampal-based novel object recognition and object place recognition behavioural tasks; (iv) restores the specific up-regulation of the activity-regulated cytoskeleton-associated protein involved in consolidation of experience-dependent synaptic plasticity; (v) relieves the loss of dendritic spine connectivity in pyramidal hippocampal CA1 neurons; (vi) rescues the Alzheimer\u2019s disease-related electrophysiological deficits in hippocampal long-term potentiation at the CA3-CA1 synapses; and (vii) mitigates the neuroinflammatory response (reactive gliosis). These findings indicate that the 20\u201322 kDa NH2-terminal tau fragment is crucial target for Alzheimer\u2019s disease therapy and prospect immunotherapy with 12A12 monoclonal antibody as safe (normal tau-preserving), beneficial approach in contrasting the early Amyloid\u3b2-dependent and independent neuropathological and cognitive alterations in affected subject

Recombinant glycine receptors: stoichiometry and kinetics.

Glycine receptors (GlyR) are anion-permeable channels that belong to the pentameric ligand-gated ion channel family. Different GlyR subtypes are known. The main synaptic form is thought to be α1β heteropentamers which mediate fast synaptic inhibition in the adult spinal cord and brainstem. Data on recombinant receptors suggest two possible stoichiometries for this subtype, 2α1:3β and 3α1:2β. Evidence for the first comes from experiments on oocytes, whereas a study in mammalian cells favours the latter, raising the possibility that stoichiometry depends on the expression system. Here, we assess the stoichiometry of α1β GlyRs in Xenopus oocytes using two different electrophysiological approaches. The first involves the use of a reporter mutation at the conserved 9΄ position of the pore-lining domain. In other receptors, this mutation shifts agonist sensitivity in proportion to the number of mutated subunits. Recordings from mutant receptors failed to point towards one or the other stoichiometry. The second approach involved single-channel recordings from conductance mutants. This approach was also inconclusive for stoichiometry. However, we provide evidence that oocytes are not a suitable expression system for the study of heteromeric glycine receptors as they are highly prone to contamination by homomers. α2 homomeric GlyRs are predominant early in development and are replaced by α1 subunits in the first postnatal days. We investigated the activation mechanism of these channels in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α2 channels can open only when all binding sites are occupied by glycine, and only after the channel undergoes a conformational change ('flip') that links binding to gating. Macroscopic data favour a two binding site model. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments when desensitization is included in the model.