Timothy Johnstone

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• Sangadi, D. K., Sangadi, A., Pláceres-Uray, F., Titus, D. J., Johnstone, T. B., Hogenkamp, D. J., Gee, K. W., & Atkins, C. M. (2024). Enhancing cognitive function in chronic TBI: The Role of α7 nicotinic acetylcholine receptor modulation. Exp Neurol, 372, 114647. doi:10.1016/j.expneurol.2023.114647
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  Traumatic brain injury (TBI) results in several pathological changes within the hippocampus that result in adverse effects on learning and memory. Therapeutic strategies to enhance learning and memory after TBI are still in the early stages of clinical development. One strategy is to target the α7 nicotinic acetylcholine receptor (nAChR), which is highly expressed in the hippocampus and contributes to the formation of long-term memory. In our previous study, we found that AVL-3288, a positive allosteric modulator of the α7 nAChR, improved cognitive recovery in rats after moderate fluid-percussion injury (FPI). However, whether AVL-3288 improved cognitive recovery specifically through the α7 nAChR was not definitively determined. In this study we utilized Chrna7 knockout mice and compared their recovery to wild-type mice treated with AVL-3288 after TBI. We hypothesized that AVL-3288 treatment would improve learning and memory in wild-type mice, but not Chrna7-/- mice after TBI. Adult male C57BL/6 wild-type and Chrna7-/- mice received sham surgery or moderate controlled cortical impact (CCI) and recovered for 3 months. Mice were then treated with vehicle or AVL-3288 at 30 min prior to contextual fear conditioning. At 3 months after CCI, expression of α7 nAChR, choline acetyltransferase (ChAT), high-affinity choline transporter (ChT), and vesicular acetylcholine transporter (VAChT) were found to be significantly decreased in the hippocampus. Treatment of wild-type mice at 3 months after CCI with AVL-3288 significantly improved cue and contextual fear conditioning, whereas no beneficial effects were observed in Chrna7-/- mice. Parietal cortex and hippocampal atrophy were not improved with AVL-3288 treatment in either wild-type or Chrna7-/- mice. Our results indicate that AVL-3288 improves cognition during the chronic recovery phase of TBI through modulation of the α7 nAChR.
• Genaro, K., Yoshimura, R. F., Doan, B., Johnstone, T. B., Hogenkamp, D. J., & Gee, K. W. (2022). Allosteric modulators of the δ GABAA receptor subtype demonstrate a therapeutic effect in morphine-antinociceptive tolerance and withdrawal in mice. Neuropharmacology, 219, 109221. doi:10.1016/j.neuropharm.2022.109221
• Koziol‐White, C., Johnstone, T. B., Corpuz, M. L., Cao, G., Orfanos, S., Parikh, V., Deeney, B., Tliba, O., Ostrom, R. S., Dainty, I., & Panettieri, R. A. (2020). Budesonide enhances agonist-induced bronchodilation in human small airways by increasing cAMP production in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol, 318(2), L345-L355. doi:10.1152/ajplung.00393.2019
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• Núñez, F., Corpuz, M. L., Fogel, D. M., Lyon, K. T., Kazarian, A. G., Johnstone, T. B., & Ostrom, R. S. (2020). Glucocorticoids rapidly stimulate cAMP production in a G_αs‐dependent manner. FASEB J, 34(2), 2882-2895. doi:10.1096/fasebj.2020.34.s1.05952
• Atkins, C. M., Chapalamadugu, M., Gee, K. W., Hogenkamp, D. J., Johnson, N. H., Johnstone, T. B., London, S. H., & Titus, D. J. (2019). Positive allosteric modulation of the α7 nicotinic acetylcholine receptor as a treatment for cognitive deficits after traumatic brain injury. PLOS ONE, 14(10), 1-20. doi:10.1371/journal.pone.0223180
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  Cognitive impairments are a common consequence of traumatic brain injury (TBI). The hippocampus is a subcortical structure that plays a key role in the formation of declarative memories and is highly vulnerable to TBI. The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in the hippocampus and reduced expression and function of this receptor are linked with cognitive impairments in Alzheimer's disease and schizophrenia. Positive allosteric modulation of α7 nAChRs with AVL-3288 enhances receptor currents and improves cognitive functioning in naïve animals and healthy human subjects. Therefore, we hypothesized that targeting the α7 nAChR with the positive allosteric modulator AVL-3288 would enhance cognitive functioning in the chronic recovery period of TBI. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury or sham surgery. At 3 months after recovery, animals were treated with vehicle or AVL-3288 at 30 min prior to cue and contextual fear conditioning and the water maze task. Treatment of TBI animals with AVL-3288 rescued learning and memory deficits in water maze retention and working memory. AVL-3288 treatment also improved cue and contextual fear memory when tested at 24 hr and 1 month after training, when TBI animals were treated acutely just during fear conditioning at 3 months post-TBI. Hippocampal atrophy but not cortical atrophy was reduced with AVL-3288 treatment in the chronic recovery phase of TBI. AVL-3288 application to acute hippocampal slices from animals at 3 months after TBI rescued basal synaptic transmission deficits and long-term potentiation (LTP) in area CA1. Our results demonstrate that AVL-3288 improves hippocampal synaptic plasticity, and learning and memory performance after TBI in the chronic recovery period. Enhancing cholinergic transmission through positive allosteric modulation of the α7 nAChR may be a novel therapeutic to improve cognition after TBI.
• Dudek, F. E., Gee, K. W., Hogenkamp, D. J., Johnstone, T. B., McCarren, H. S., McDonough, J. H., & Spampanato, J. (2019). Enaminone Modulators of Extrasynaptic α4β3δ γ-Aminobutyric AcidA Receptors Reverse Electrographic Status Epilepticus in the Rat After Acute Organophosphorus Poisoning. Frontiers in Pharmacology, 10, 560. doi:10.3389/fphar.2019.00560
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  Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic gamma-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 hr (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 hr) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication.
• Koziol‐White, C., Johnstone, T. B., Ostrom, R. S., & Panettieri, R. A. (2019). Budesonide Augments Receptor-Dependent and -Independent Bronchodilation of Human Small Airways and Localized cAMP Production in Human Airway Smooth Muscle. Am J Physiol Lung Cell Mol Physiol, 318(2), L345-L355. doi:10.1164/ajrccm-conference.2019.199.1_meetingabstracts.a2841
• Carver, C. M., Gee, K. W., Hogenkamp, D. J., Johnstone, T. B., Ramanathan, G., Reddy, D. S., & Yoshimura, R. F. (2018). Role of β 2/3 -specific GABA-A receptor isoforms in the development of hippocampus kindling epileptogenesis. Epilepsy & Behavior, 82, 57-63. doi:10.1016/j.yebeh.2018.02.020
• Johnstone, T. B., Smith, K. H., Koziol‐White, C., Li, F., Kazarian, A. G., Corpuz, M. L., Shumyatcher, M., Ehlert, F. J., Himes, B. E., Panettieri, R. A., & Ostrom, R. S. (2018). PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by β₂-Adrenergic Receptors and Adenylyl Cyclase 6. Am J Respir Cell Mol Biol, 58(4), 530-541. doi:10.1165/rcmb.2017-0294oc
• Johnstone, T. B., Xie, J. Y., Qu, C., Wasiak, D. J., Hogenkamp, D. J., Porreca, F., & Gee, K. W. (2018). Positive allosteric modulators of nonbenzodiazepine γ-aminobutyric acidA receptor subtypes for the treatment of chronic pain. Pain, 160(1), 198-209. doi:10.1097/j.pain.0000000000001392
• Edmonds, S. A., Freedman, R., Gee, K. W., Harris, J. G., Hogenkamp, D. J., Johnson, L., Johnstone, T. B., Kanner, R. E., Olincy, A., Sauer, W. H., Tran, M. B., & Yoshimura, R. F. (2017). First in human trial of a type I positive allosteric modulator of alpha7-nicotinic acetylcholine receptors: Pharmacokinetics, safety, and evidence for neurocognitive effect of AVL-3288. Journal of Psychopharmacology, 31(4), 434-441. doi:10.1177/0269881117691590
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  Type I positive allosteric modulators (PAMs) of the alpha7-nicotinic receptor enhance its cholinergic activation while preserving the spatiotemporal features of synaptic transmission and the receptor’s characteristic rapid desensitization kinetics. Alpha7-nicotinic receptor agonists have shown promise for improving cognition in schizophrenia, but longer-term trials have been disappointing. Therefore, the type I PAM AVL-3288 was evaluated for safety and preliminary evidence of neurocognitive effect in healthy human subjects. Single-dose oral administration in ascending doses was conducted in a double-blind, placebo-controlled Phase I trial in non-smokers. The trial found indication of positive but non-significant effects on neurocognition at 10 and 30 mg, two doses that produced overlapping peak levels. There was also some evidence for effects on inhibition of the P50 auditory evoked potential to repeated stimuli, a biomarker that responds to alpha7-nicotinic receptor activation. The pharmacokinetic characteristics were consistent between subjects, and there were no safety concerns. The effects and safety profile were also assessed at 3 mg in a cohort of smokers, in whom concurrent nicotine administration did not alter either effects or safety. The trial demonstrates that a type I PAM can be safely administered to humans and that it has potential positive neurocognitive effects in central nervous system (CNS) disorders.
• Johnstone, T. B., Agarwal, S., Harvey, R. D., & Ostrom, R. S. (2017). cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes. Molecular Pharmacology, 93(4), 270-276. doi:10.1124/mol.117.110825
• Yoshimura, R. F., Tran, M., Hogenkamp, D. J., Ayala, N. L., Johnstone, T. B., Dunnigan, A., Gee, T. K., & Gee, K. W. (2017). Allosteric modulation of nicotinic and GABA A receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model. Neuropharmacology, 126, 38-47. doi:10.1016/j.neuropharm.2017.08.029
• Gee, K. W., Hogenkamp, D. J., Johnstone, T. B., Kanner, R. E., Tran, M. B., & Yoshimura, R. F. (2014). Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid. Psychopharmacology, 231(7), 3517-3524. doi:10.1007/s00213-014-3494-5
• Bascom, G. D., Ford-Hutchinson, T. A., Gee, K. W., Hogenkamp, D. J., Johnstone, T. B., Li, W., Lu, L., Rollins, H., Tran, M. B., Whittemore, E. R., & Yoshimura, R. F. (2013). Design, Synthesis, and Activity of a Series of Arylpyrid-3-ylmethanones as Type I Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors. Journal of Medicinal Chemistry, 56(21), 8352-8365. doi:10.1021/jm400704g
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  A series of novel arylpyrid-3-ylmethanones (7a-aa) were designed as modulators of α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be type I positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes. Structure-activity relationship (SAR) studies resulted in the identification of compound 7v as a potent and efficacious type I PAM with maximum modulation of a nicotine EC5 response of 1200% and EC50 = 0.18 μM. Compound 7z was active in reversing the effect of scopolamine in the novel object recognition (NOR) paradigm with a minimum effective ip dose of 1.0 mg/kg (2.7 μmol/kg). This effect was blocked by the selective α7 nAChR antagonist methyllycaconitine (MLA). These compounds are potent type I positive allosteric modulators of α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive deficits in schizophrenia and Alzheimer's disease.
• Yoshimura, R. F., Tran, M., Hogenkamp, D. J., Johnstone, T. B., Xie, J. Y., Porreca, F., & Gee, K. W. (2013). Limited central side effects of a β-subunit subtype-selective GABA_A receptor allosteric modulator. J Psychopharmacology, 28(5), 472-478. doi:10.1177/0269881113507643
• Belluzzi, J. D., Gee, K. W., Gu, Z., Hogenkamp, D. J., Huang, J., Johnstone, T. B., Tran, M. B., Villégier, A., Whittemore, E. R., Yakel, J. L., & Yoshimura, R. F. (2011). Allosteric Modulation of Related Ligand-Gated Ion Channels Synergistically Induces Long-Term Potentiation in the Hippocampus and Enhances Cognition. Journal of Pharmacology and Experimental Therapeutics, 336(3), 908-915. doi:10.1124/jpet.110.176255
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  α5 Subunit-containing GABA(A) receptors (GABA(A)Rs) and α7 neuronal nicotinic-acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels (LGICs) that mediate cognitive and attentional processes in the hippocampus. α5 GABA(A)Rs alter network activity by tonic inhibition of CA1/CA3 pyramidal cells of the hippocampus. Postsynaptic α7 nAChRs in the hippocampus regulate inhibitory GABAergic interneuron activity required for synchronization of pyramidal neurons in the CA1, whereas presynaptic α7 nAChRs regulate glutamate release. Can simultaneous allosteric modulation of these LGICs produce synergistic effects on cognition? We show that combined transient application of two allosteric modulators that individually 1) inhibit α5 GABA(A)Rs and 2) enhance α7 nAChRs causes long-term potentiation (LTP) of mossy fiber stimulation-induced excitatory postsynaptic currents (EPSC) from CA1 pyramidal neurons of rat hippocampal slices. The LTP effect evoked by two compounds is replicated by 3-(2,5-difluorophenyl)-6-(N-ethylindol-5-yl)-1,2,4-triazolo[4,3-b]pyridazine (522-054), a compound we designed to simultaneously inhibit α5 GABA(A)Rs and enhance α7 nAChRs. Selective antagonists for either receptor block sustained EPSC potentiation produced by 522-054. In vivo, 522-054 enhances performance in the radial arm maze and facilitates attentional states in the five-choice serial reaction time trial with similar receptor antagonist sensitivity. These observations may translate into therapeutic utility of dual action compounds in diseases of hippocampal-based cognitive impairment.
• Bagnera, R. E., Belluzzi, J. D., Gee, K. W., Hogenkamp, D. J., Huang, J., Johnstone, T. B., Tran, M. B., Whittemore, E. R., & Yoshimura, R. F. (2010). Limiting Activity at β₁-Subunit-Containing GABA_A Receptor Subtypes Reduces Ataxia. Journal of Pharmacology and Experimental Therapeutics, 332(3), 1040-1053. doi:10.1124/jpet.109.161885
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  GABA(A) receptor (R) positive allosteric modulators that selectively modulate GABA(A)Rs containing beta(2)- and/or beta(3)- over beta(1)-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,"beta(2/3)-selective" GABA(A)R positive allosteric modulators are nonbenzodiazepines (nonBZs), do not show alpha-subunit isoform selectivity, yet have anxiolytic efficacy with reduced ataxic/sedative effects in animal models and humans. Here, we report on an enantiomeric pair of nonBZ GABA(A)R positive allosteric modulators that demonstrate differential beta-subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other beta(2/3)-subunit selective, alpha-subunit isoform-selective, BZ and nonBZ GABA(A) positive allosteric modulators using electrophysiological, pharmacokinetic, and behavioral assays to test the hypothesis that ataxia may be correlated with the extent of modulation at beta(1)-subunit-containing GABA(A)Rs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABA(A)R with BZ-like anxiolytic efficacy by reducing or eliminating activity at beta(1)-subunit-containing GABA(A)Rs.
• Bagnera, R. E., Gee, K. W., Hogenkamp, D. J., Huang, J., Johnstone, T. B., Li, W., Tran, M. B., & Whittemore, E. R. (2007). Enaminone Amides as Novel Orally Active GABA_A Receptor Modulators. Journal of Medicinal Chemistry, 50(14), 3369-3379. doi:10.1021/jm070083v
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  A series of enaminone esters and amides have been developed as potent allosteric modulators of γ-aminobutyric acidA (GABAA) receptors. The compounds bind to a novel modulatory site that is independent of the benzodiazepine (BZ), isosteric GABA, and neuroactive steroid binding sites. Structure−activity relationship (SAR) studies resulted in the synthesis of the c-Bu amide 16h with an in vitro potency of 7 nM based on inhibition of [35S]TBPS binding. The activity of the enaminones as positive allosteric modulators was confirmed with electrophysiological measurements in oocytes expressing α1β2γ2L GABAA receptors. The i-Pr, s-Bu, c-Pr, and c-Bu amides (16e−h) were orally active in mice with profound central nervous system depressant effects. The i-Pr amide 16e was an orally active anxiolytic in the mouse light−dark paradigm.
• Broide, R. S., Gee, K. W., Hogenkamp, D. J., Johnstone, T. B., Ng, H. J., Stevens, K. E., Tran, M. B., Whittemore, E. R., & Zheng, L. (2007). Nootropic α7 nicotinic receptor allosteric modulator derived from GABA _A receptor modulators. Proceedings of the National Academy of Sciences of the United States of America, 104(19), 8059-8064. doi:10.1073/pnas.0701321104
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  Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.
• Wang, J., Hogenkamp, D. J., Tran, M., Li, W., Yoshimura, R. F., Johnstone, T. B., Shen, W., & Gee, K. W. (2006). Reversible lipidization for the oral delivery of leu-enkephalin. J Drug Target, 14(3), 127-136. doi:10.1080/10611860600648221
• Coyne, L., Gee, K. W., Halliwell, R. F., Hogenkamp, D. J., Johnstone, T. B., Li, W., Su, J., Tran, M. B., Wang, J., & Yoshimura, R. F. (2004). Modifying quinolone antibiotics yields new anxiolytics. Nature Medicine, 10(1), 31-32. doi:10.1038/nm967