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Prof. Uri Littauer Lecture: Changes in the brain during chronic exposure to nicotine Studies on

Prof. Uri Littauer Lecture: Changes in the brain during chronic exposure to nicotine Studies on genes, receptor binding, proteins, drugs, cells, circuits, and behavior. Henry Lester. February, 2009. There are excellent reasons to focus on

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Prof. Uri Littauer Lecture: Changes in the brain during chronic exposure to nicotine Studies on

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  1. Prof. Uri Littauer Lecture: Changes in the brain during chronic exposure to nicotine Studies on genes, receptor binding, proteins, drugs, cells, circuits, and behavior Henry Lester February, 2009

  2. There are excellent reasons to focus on the nicotinic acetylcholine receptors (nAChR) themselves: subtypes: a1- a10, 1 - 4, γ, δ, ε There are good reasons to focus on a42* nAChRs Conclusions from hypersensitive and knockout mice (2005): Activation of a4-containing (a4b2*) receptors by nicotine Is sufficient and necessary for tolerance, sensitization, reward, (but withdrawal?) Picciotto, Marubio, Maskos, Tapper . . . . Caltech, Pasteur What are the mechanisms? (But remember that some a42* receptors contain: ≥ one 5, 6, or β3 subunit)

  3. Nicotine and ACh act on many of the same receptors, but . . . • 1. Nicotine is highly membrane-permeant. ACh is not. • Ratio unknown, probably > 1000. • 2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~104 /s.) In mouse, nicotine is eliminated with a half time of ~ 10 min. • Ratio: ~105 • EC50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM. • Ratio, ~10. Justified to square this because nH = 2. • Functional ratio, ~100. What causes this difference?

  4. Nearly Complete Nicotinic Acetylcholine Receptor, a Well-Studied Cys-loop Receptor ~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 106 Binding region Membrane region Colored by secondary structure Colored by subunit (chain) Cytosolic region (incomplete)

  5. The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004) aY198 C2 aW149 B aY93 A aY190 C1 non-aW55 D (Muscle Nicotinic numbering)

  6. WT WT, without cation-π interaction Nicotine makes a stronger cation-π interaction with Trp B at α4β2 receptors than at muscle receptors; this partially explains α4β2 receptors’ high binding affinity for nicotine.

  7. Nicotine makes a stronger H-bond to a backbone carbonyl at α4β2 than at muscle receptors:With amide to ester substitution,EC50 increases 20-fold vs 1.5-fold Weaker hydrogen bond C2 B A C1 Deleted hydrogen bond D

  8. Nicotine EC50 values: Muscle nAChR single component ~ 400 μM α4β2 two components ~ 1 μM, ~200 μM Underlying the 400-fold higher nicotine sensitivity of neuronal vs muscle receptors: Factor of ~16 for the cation-π interaction; Factor of ~ 12 for H-bond; 16 x 12 = 192. We still can’t explain a factor of 400/192 ~ 2. Xiu, Puskar, Shanata, Lester, Dougherty. 2009. Nature in press

  9. receptor G protein i q s t kinase effector channel enzyme intracellular messenger cAMP Ca2+ phosphorylated protein Brunzell, Russell, & Piccotto, 2003 Possible molecular mechanism #1 for changes with chronic nicotine: Signal transduction triggered by a ligand-gated channel NMDA receptors and nAChRs are highly permeable to Ca2+ as well as to Na+.

  10. Possible Mechanism #2 for changes with chronic nicotine: “Upregulation” Chronic exposure to nicotine causes upregulation of nicotinic receptor binding (1983: Marks & Collins; Schwartz and Kellar); Upregulation 1) Involves no change in receptor mRNA level; 2) Depends on subunit composition (Lindstrom, Kellar, Perry). Shown in experiments on clonal cell lines transfected with nAChR subunits: Nicotine seems to act as a “pharmacological chaperone” (Lukas, Lindstrom) or “maturational enhancer” (Sallette, Changeux, & Corringer; Heinemann) or “Novel slow stabilizer” (Green). Upregulation is “cell autonomous” and “receptor autonomous” (Henry).

  11. Nicotine accumulates in acidic cells & organelles Binding eventually favors high-affinity states Bound states with increasing affinity unbound + + 1 mM Nicotine+ (pKa = 7.8) + C Highest affinity bound state AC pH 7.4 pH 7.0 Free Energy A2C A2O A2D Reaction Coordinate 2.5 mM Nicotine+ Nicotine stabilizes subunit interfaces ? Covalently stabilized AR*HS + nicotine RHS RLS Increasingly stable assembled states Degradation Free subunits Nicotine Increased High-Sensitivity Receptors Free Energy Reaction Coordinate hr 0 20 40 60 Upregulation is a thermodynamic consequence of nicotine-nAChR interactions SePhaCHARNS “Selective Pharmacological Chaperoning of Acetylcholine Receptor Number and Stoichiometry”

  12. High-resolution fluorescence microscopy to study SePhaChARNS LTP / Opioids: regulation starts here TIRFM PM ER Pharmacological chaperoning: upregulation starts here FRET Golgi Nucleus

  13. M3 - M4 α4 N C N C loop M3 - M4 loop Ligand binding M1 M2 M3 M4 M4 HA tag XFP c - myc tag XFP b a 2 - XFP 4 - XFP FRET pairs (m = monomeric) EYFP XFP = mCerulean ECFP mEGFP mVenus mCherry mEYFP Förster resonance energy transfer (FRET): a test for subunit proximity β2

  14. 50% α-CFP, 50% α-YFP 1/4 1/4 1/2 E No FRET b/a =1.62; 1.62-6 = 0.055 1/8 1/8 1/8 100% α3β2 E1 E2 E3 E4 100% α2β3 No FRET 1/8 1/4 1/4 % receptors with α3 Theory of FRET in pentameric receptors with αnβ(5-n)subunits

  15. A key experiment: changes in subunit stoichiometry caused by chronic nicotine! Neuro2a Cagdas Son

  16. 4 hour nicotine exposure causes an increase in (a4)2(b2)3 assembly WHOLE CELL + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.5 ± 0.18 w1 = 2.4 ± 0.1 A1 = 130438 ± 36122 xc2 = 10.1 ± 0.26 w2 = 2.24 ± 0.14 A2 = 64907 ± 26106 WHOLE CELL No treatment R2 = 0.999 y0 = 0 xc1 = 8.7 ± 0.06 w1 = 2.22 ± 0.12 A1 = 88465 ± 34150 xc2 = 10 ± 0.36 w2 = 2.92 ± 0.19 A2 = 109476 ± 34316 GOLGI No treatment R2 = 0.999 y0 = 0 xc1 = 8.28 ± 0.07 w1 = 1.9 ± 0.05 A1 = 6756 ± 620 xc2 = 9.72 ± 0.07 w2 = 1.8 ± 0.04 A2 = 5298 ± 621 GOLGI + 1 mM NICOTINE 4 h R2 = 0.998 y0 = 0 xc1 = 8.37 ± 0.02 w1 = 2.33 ± 0.03 A1 = 11498 ± 239 xc2 = 10.21 ± 0.04 w2 = 1.51 ± 0.06 A2 = 1986 ± 233

  17. Total Internal Reflection Fluorescence Microscopy (TIRFM)

  18. Differential subcellular localization and dynamics of α4GFP* receptors plasma memb. mCherry overlay α4GFPβ2 overlay α4GFPβ4(1:1) α4GFPβ2 3 RXR per β subunit α4GFPβ4 0 RXR per β subunit

  19. Leu9’Ala-YFP, YFP, CFP Strategy to evaluate the cell specificity of a4* upregulation in chronic nicotine 1. Generate knock-in mice with fully functional, fluorescent a4* receptors 2. Expose the mice to chronic nicotine 3. Find the brain regions and cell types with changed receptor levels 4. Perform physiological experiments on these regions and cells to verify function 5. Model the cellular and circuit changes

  20. Cellular and subcellular specificity of SePhaChARNS CA EC MH DG IPN Medial Perforant Path Striatum SNc Thalamus, superior colliculus SNr * = upregulation shown with electrophysiology Nashmi et al J Neurosci 2007; Xiao et al, in prep.

  21. The Caltech a4 fluorescent mice . . . normal in all respects

  22. DA neuron, ~ 1700 spikes 4*, 6*, and/or 7 6 Nicotine injection Frequency, Hz 4 2 A B C D VTA 0.05 mV 2 ms DAergic 0 25 4* only GABAergic 20 0.1 mV Frequency, Hz 0.5 ms 15 10 V GABAergic neuron (5 s smoothing), ~ 8300 spikes 5 0 100 200 300 400 500 600 700 0 s VTA GABAergic and DA neurons have contrasting responses to nicotine in vivo WT mouse

  23. a4-YFP knock-in: substantia nigra pars compacta neurons Spectrally unmixed background autofluorescence Spectrally unmixed a4YFP 10 mm 10 mm Raad Nashmi

  24. Midbrain data show cell specificity of SePhaChARNS Chronic nicotine does not change a4 levels in dopaminergic neurons . . . Substantia Nigra Pars Compacta (& VTA, not shown) α4 intensity per TH+ neuron . . . but does upregulate a4 levels in GABAergic inhibitory neurons. Substantia Nigra Pars Reticulata (& VTA, not shown) α4 intensity per GAD+ neuron

  25. Chronic Nicotine Tolerance Endogenous ACh Upregulated a4* nAChRs 2A Craving 1A Reward Endogenous ACh 4.0 Yoked saline 3.5 Yoked nicotine Decreased Reward 3.0 Plus Acute Nicotine (1st expsoure) 2.5 2B Dialysate DA (nM) 2.0 1B 1.5 Plus Acute Nicotine (repeated exposure) 1.0 0.5 Saline Nicotine 0.0 0 20 40 60 80 120 140 160 180 -40 -20 100 + acute nicotine Time (min) 2A 1A 1B 2B Chronic nicotine cell-specifically up-regulates functional a4* receptors: Basis for circuit-based tolerance in midbrain (Nashmi et al, 2007) Chronic Saline Endogenous ACh VTA NAc LDT DAergic Cholinergic GABAergic Rahman et al, 2004

  26. Midbrain slice recordings: functional upregulated receptors in a simple circuit In SNr of α4 knockout, these effects of chronic nicotine vanish Cheng Xiao

  27. V Chronic nicotine increases firing rate of SNr GABAergic neurons in vivo . . . . . . we’re still gathering data for DA neurons Cheng Xiao

  28. Chronic nicotine causes cognitive sensitization In the human context, cognitive sensitization is epitomized by smokers’ reports that they think better when they smoke; this anecdotal observation is confirmed by data that smokers who smoke nicotine cigarettes (but not nicotine-free cigarettes) display certain cognitive enhancements (Rusted and Warburton, 1992; Rusted et al., 1995). In the rodent context, mice show more contextual fear conditioning if, one day after withdrawal from chronic nicotine, they receive an acute nicotine dose (Davis et al., 2005); β* dependent also chronic nicotine produces better spatial working memory performance in the radial arm maze (Levin et al., 1990; Levin et al., 1996).

  29. Chronic nicotine increases perforant path a4 fluorescence ~ 2-fold Alveus Py Or Rad • TV Bliss, T Lömo (1973) • Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. • J Physiol. 232:331-56. LMol 200 mm Temperoammonic Path Medial Perforant Path

  30. Acute Nicotine Acute Saline 80 min 10 min 80 min 10 min Chronic Chronic 1 mV Saline Saline 10 ms 0.5 mV 0.5 mV 10 ms 5 ms Nicotine 1mV Nicotine 1 mV 10 ms 10 ms Simple model for cognitive sensitization: chronic nicotine + acute nicotine lowers the threshold for perforant pathway LTP Acute Nicotine Acute Saline Chronic Nicotine Acute Chronic Chronic

  31. Some changes in the brain during chronic exposure to nicotine 1. Nicotine potently activates some neuronal nAChRs (because it participates in both cation-π and H-bond interactions within the conserved aromatic box). Genes Binding Proteins Cells Circuits Behavior • Chronic exposure to nicotine chaperones α4β2* number and stoichiometry. 3. These processes lead to cell-specific α4β2*upregulation. • a. Upregulation explains tolerance to chronic nicotine, via a GABAergic-DA circuit in the midbrain. • b. Upregulation also explains enhanced LTP in the perforant path, via a direct presynaptic mechanism. This is a simple model for cognitive sensitization 5. We do not yet understand several processes, including somatic signs of withdrawal and stress-induced nicotine use.

  32. Understanding the changes in the brain during chronic exposure to nicotine: Translational relevance of SePhaChARNS • Nicotine addiction • (~1 billion people, since 1540) • Strong inverse correlation between smoking & Parkinson’s disease • (~ 50 million people, since 1819) • Tobacco use suppresses seizures in patients with autosomal dominant nocturnal frontal-lobe epilepsy, linked to the α4 and β2 subunits • (~1000 patients, since 1994)

  33. Dennis Dougherty Kiowa Bower, Shawna Frazier, Ariel Hanek, Fraser Moss, Nyssa Puskar, Rigo Pantoja, Kristin Rule, Erik Rodriguez, Jai Shanata, Mike Torrice, Joanne Xiu Neil Harrison, Sarah Lummis, Claire Padgett, Kerry Price, Andy Thompson Stephan Pless, Joe Lynch Caltech “Unnatural Club” Univ. of Cambridge Uni Queensland Caltech “Alpha Club” Bruce Cohen, Purnima Deshpande, Ryan Drenan, Carlos Fonck, Sheri McKinney, Raad Nashmi, Johannes Schwarz, Rahul Srinivasan, Cagdas Son, Andrew Tapper, Larry Wade, Cheng Xiao Al Collins, Sharon Grady, Mike Marks, Erin Meyers, Tristan McClure-Begley, Charles Wageman, Paul Whiteaker Merouane Bencherif, Greg Gatto, Daniel Yohannes Jon Lindstrom Mike McIntosh Julie Miwa, Nathaniel Heintz Univ of Colorado, Boulder Targacept Univ. Pennsylvania Univ. Utah Rockefeller Univ

  34. a P P P g b Activated GPCRs are sometimes phosphorylated and endocytosed. This “downregulation” terminates signalling. During activation, the G protein leaves . . . P . . . revealing phosphorylation sites . . . . . .other proteins bind to the phosphates . . . kinase But continual signalling can activate genes (not a synaptic vesicle) . . . triggering endocytosis.

  35. FRET with fluorescent subunits tells us about nicotinic receptor assembly * a4/b2Y/b2C a4Y/a4C/b2 a6Y/a6C/b2 a4/b2/b3Y/b3C Neuro2a

  36. 1 million channels nicotine 20 seconds The nicotine video Produced for Pfizer to explain varenicline (Chantix) to physicians This summarizes knowledge in ~ 2004. “physical” addiction vs “psychological” addiction. Desensitization and “Upregulation” Some abbreviations on future slides: ACh, acetylcholine nAChR, nicotinic acetylcholine receptor DA, dopamine

  37. Chronic nicotine causes tolerance of dopamine release 4.0 Yoked saline 3.5 Yoked nicotine 3.0 2.5 Dialysate DA (nM) 2.0 1.5 1.0 0.5 Saline Nicotine 0.0 Yoked animal 0 40 80 120 160 -40 Master animal Time (min) Rahman, Zhang, Engleman, & Corrigall, 2004

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