1. Changes in the Brain during Chronic Exposure to Nicotine September 2010 Nicotine Addiction Nicotine Addiction Parkinson’s Disease Parkinson’s Disease ADNFLE Behavior Circuits Synapses Neurons Intracell. Binding Nic vs ACh Proteins RNA Genes Today’s focus: Inadvertent therapeutic effect of chronic nicotine Three mechanistic hypotheses 1.Circuit consequences of cell-specific upregulation 2.Axon terminals of DA neurons 3.Pharmacological chaperoning in ER 1

2. 1 st and 2 nd hypotheses. Cellular and axon/soma specificity of SePhaChARNS: α4* nAChRs Thalamus, superior colliculus SNc, VTA SNr, VTA Striatum Upregulation? TransmitterSomaTerm.Region / projection Glu??Yes Entorhinal cortex → dentate gyrus AChNo Medial habenula → Interpeduncular nucleus DANoYes Ventral tegmental area, substantia nigra pars compacta → Striatum GABAAYes SN pars reticulata, VTA → SNC, VTA CA DG EC Medial Perforant Path Nashmi et al J Neurosci 2007; Xiao et al, J. Neurosci 2009 2 MH IPN

3. GABAergic neurons have increased (or more regular?) firing in chronic nicotine... Thalamus, superior colliculus GABAergic DAergic SNc SNr Endogenous ACh PPTg Cholinergic Striatum Upregulated a4* nAChRs... Analogous to “deep brain stimulation” in subthalamic nucleus? STN 1 st Hypothesis for PD neuroprotection by Chronic nicotine: Circuit-based mechanism in substantia nigra via Cholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain 3

4. 4 4 α4-eGFP β2 α4-eGFP β4 β2 subunits govern the ER localization of α4β2 nAChRs 5 µm

5. 5 5 LFMAAQA 10 µm Nicotine and mutant β2 subunits overcome a rate limiting ER exit step in α4β2 nAChR trafficking to the PM Mutations overcome a rate limiting step in ER export Nicotine upregulates nAChRs by a distinct mechanism

6. Golgi complex Clathrin Secretory vesicle COPI Early endosome COPI ERGIC COPII nAChR Plasma membrane Endoplasmic reticulum nAChR 6 “Endoplasmic reticulum stress” occurs when the cell cannot clear newly synthesized proteins from the ER COP II: Sec23/24 heterodimer GTPase: Sar1 scission ER lumen ER membrane Cytosolic compartment Mancias & Goldberg, Traffic 2005

7. 7 7 3 rd hypothesis: Nicotine and mutant nAChRs increase ER exit sites 10 µm M3-M4 mutations increase ER exit of nAChRs Nicotine exposure increases ER exit of wt nAChRs Upregulation is initiated prior to ER exit of nAChRs

8. 8 8 1.06 2.343.8 2.70.574.83 4.62.26 1.72.872.32.05 Nicotine and mutant receptors alter nAChR stoichiometry in the ER

9. 9 9 Nicotine and DM receptors alter trans-Golgi network activity α4-eGFP β2-wt, No Nic GalT-mcherry, No Nic GalT-mcherryα4-eGFP β2-DMMerge α4-eGFP β2-wt + 0.1 µM Nic GalT-mcherry + 0.1 µM Nic

10. Specific Expression of  6 nAChRs in Midbrain DA Neurons 10 WT  6 L9′S  4 L9′A DA NeuronsGABA Neurons

11.  4 Subunits are Required for Behavioral Hyperactivity Observed in  6 L9’S Mice Ryan Drenan, Sheri McKinney 11 Drenan et al., in preparation 2010

12. Conclusion:  4 Subunits are Critical to  6 nAChR Function In Vivo 12

13. 2 nd hypotheses (DA terminals). α4* nAChRs on dopaminergic terminals exert a tonic inhibition of glutamate release. In chronic nicotine, this tonic inhibition is greater because of upregulated α4* nAChRs This could be neuroprotective (Xiao et al, 2009). 13

14. A B C Vm = -60 mV, 0.3 mM ACh (0.1 s puff) 15 ms 3 pA Amplitude (pA) -2.0-1.5-0.5 0 20 40 60 Vm = -60 mV 1.44 pA → 24 pS Counts Amplitude (pA) -3.0-2.5-2.0-1.5 0 10 20 30 Vm = -90 mV 1.93 pA → 21.4 pS D Reliable α6β2 expression in N2a cells 14

15. 15 Chronic nicotine exposure downregulates PM α6-meGFP β2 nAChRs ER subtracted No Nic + 0.1 µM Nic ER subtracted

16. Forms receptors with α3β4 and α4β2 subunits Not thought to contribute to ligand binding interface Smallest intracellular loop (~70aa) Regulated at the level of brain region, cell type and possibly mRNA GWAS and candidate gene studies identified a SNP in the gene Chrna5. This Aspartic Acid to Asparagine mutation at position 398 appears to confer an increased risk for nicotine addiction (D398N) KO mice show differences in nicotine self administration compared to wt mice α5* receptors are not thought to upregulate when exposed to nicotine 16

17. Two stoichiometries of α4β2 receptors One stoichiometry of α4β2α5 receptors? α4α4 α4α4 β2β2 17 α4α4 β2β2 α4α4 α4α4 β2β2 α4α4 α4α4 β2β2 β2β2 β2β2 α5α5 β2β2

18. 18 HEK 293 1. α4GFPβ2 2. α4GFPβ2 α5stitzel 3. α4β2 α5stitzel GFP385 4. α4β2 α5stitzel GFP385L 5. α4β2 α5stitzel GFP378L 6. α4β2 α5-IDT GFP385L 7. α4β2 α5-IDT GFP378L 8. α4β2 α5-IDT C-Term GFP 9. α4β2 α5-IDT D/N C-Term GFP 10. α4β2 β3P379 YFP 500ng ea. 1 2 34 7 6 5 8 910

19. Moving Forward with α 5... Transfection of α5-GFP into HEK293 cells – NFRET studies yield information about receptor assembly – TIRF studies yield information about surface trafficking Transfection of α5-GFP into mouse neurons – Live and fixed cell confocal imaging of α5-GFP localization in neurons All experiments will compare α5-GFP and α5D398N-GFP under + and - nicotine conditions for changes in receptor behavior 19

20. Lynx proteins modulate (inhibit) nicotinic receptor function 1-2 Lynx proteins are GPI-linked variants of  -neurotoxins (e.g.  -bungarotoxin) 1 Removal of lynx using genetically engineered mice cause nicotinic receptor hypsersensitivity and enhanced learning and memory 3 lynx  btx 1.Miwa et al., Neuron, 1999 2.Ibanez-Tallon et al., Neuron, 2002 3. Miwa et al., Neuron, 2006 20

21.  btx and AChBP  -bungarotoxin binds to nAChRs at subunit interfaces, suggesting a possible site of action of lynx on the receptor. lynx proteins are GPI-linked membrane proteins. GPI-linked protein sort to specialized lipid domains (e.g. rafts). Lipid rafts have been shown to stabilize nicotinic receptors within synaptic structures, and reduce receptor mobility. Therefore, lynx:nAChR interactions may cause receptors to localize to specialized domains and reduce receptor mobility. 21

22. Questions: Do nAChR co-localize with lipid rafts? Labeling rafts by incubating transfected cells with labeled cholera toxin 1) co-labeling:  4  2 receptors fused in-frame to gfp/cherry 2) co-labeling:  7 receptors with labeled  -bungarotoxin Does disruption of lipid rafts increase receptor mobility? Image cells with transfected receptor (in-frame labeled). Deplete cholesterol in rafts with methyl-  –cyclodextran) Does removal of lynx (e.g. lynx1KO mice) alter the mobility, trafficking pathways, or final localization, of nAChRs? Image primary neuronal cultures from wt vs. ko mice, transfected with labeled receptor 22

23. 23 Differential effects of nicotinic ligands on the assembly, ER exit and PM localization of nAChRs NFRET using α4-mcherry + β2-eGFP Nicotine assembles (α4) 2 (β2) 3 nAChRs Cytisine assembles (α4) 3 (β2) 2 nAChRs Both drugs affect assembly in the ER

24. 24 Differential effects of nicotinic ligands on the assembly, ER exit and PM localization of nAChRs  4-mCherry +  2-eGFP Sec24D-eGFP Merge No Drug + 0.1 µM Nic, 48 h + 0.1 µM Cyt, 48 h 10 µm

25. 25 Differential effects of nicotinic ligands on the assembly, ER exit and PM localization of nAChRs No Drug + 0.1 µM Nic, 48 h 10 µm  4-mCherry +  2-eGFP Sec24D-eGFP Merge