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Changes in the Brain during Chronic Exposure to Nicotine PowerPoint PPT Presentation


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Behavior. September 2010. Changes in the Brain during Chronic Exposure to Nicotine. Circuits. Synapses. Neurons. Nicotine Addiction. Intracell. Binding. Parkinson’s Disease. Today’s focus: Inadvertent therapeutic effect of chronic nicotine Three mechanistic hypotheses

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Changes in the Brain during Chronic Exposure to Nicotine

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Changes in the brain during chronic exposure to nicotine

Behavior

September 2010

Changes in the Brain during Chronic Exposure to Nicotine

Circuits

Synapses

Neurons

Nicotine

Addiction

Intracell.

Binding

Parkinson’s

Disease

  • Today’s focus:

  • Inadvertent therapeutic effect of chronic nicotine

  • Three mechanistic hypotheses

  • Circuit consequences of cell-specific upregulation

  • Axon terminals of DA neurons

  • Pharmacological chaperoning in ER

Nic vs ACh

ADNFLE

Proteins

RNA

Genes


Changes in the brain during chronic exposure to nicotine

1st and 2nd hypotheses.

Cellular and axon/soma specificity of SePhaChARNS: α4* nAChRs

CA

EC

MH

DG

IPN

Medial Perforant Path

Striatum

SNc, VTA

Thalamus,

superior

colliculus

SNr,

VTA

Nashmi et al J Neurosci 2007; Xiao et al, J. Neurosci 2009


Changes in the brain during chronic exposure to nicotine

1st Hypothesis for PD neuroprotection by Chronic nicotine:

Circuit-based mechanism in substantia nigra

via

Cholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain

. . . Analogous to

“deep brain stimulation” in subthalamic nucleus?

STN

Striatum

SNc

DAergic

PPTg

GABAergic neurons

have increased

(or more regular?) firing

in chronic nicotine. . .

Thalamus,

superior

colliculus

Cholinergic

GABAergic

SNr

Endogenous ACh

Upregulated a4* nAChRs


Changes in the brain during chronic exposure to nicotine

β2 subunits govern the ER localization of α4β2 nAChRs

α4-eGFP β2

5 µm

α4-eGFP β4

4


Changes in the brain during chronic exposure to nicotine

Nicotine and mutant β2 subunits overcome a rate limiting ER exit step in α4β2 nAChR trafficking to the PM

LFM

AAQA

10 µm

  • Mutations overcome a rate limiting step in ER export

  • Nicotine upregulates nAChRs by a distinct mechanism

5


Changes in the brain during chronic exposure to nicotine

“Endoplasmic reticulum stress” occurs when the cell cannot clear newly synthesized proteins from the ER

GTPase: Sar1

COP II: Sec23/24

heterodimer

scission

ER lumen

ER membrane

Cytosolic compartment

Golgi

complex

Endoplasmic

reticulum

Plasma

membrane

Early

endosome

COPI

ERGIC

Clathrin

COPII

COPI

nAChR

nAChR

Secretory vesicle

Mancias &

Goldberg,

Traffic 2005


Changes in the brain during chronic exposure to nicotine

3rd 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

7


Changes in the brain during chronic exposure to nicotine

Nicotine and mutant receptors alter nAChR stoichiometry in the ER

1.06

2.7

0.57

4.83

2.34

3.8

2.26

4.6

2.05

2.3

1.7

2.87

8


Changes in the brain during chronic exposure to nicotine

Nicotine and DM receptors alter trans-Golgi network activity

GalT-mcherry, No Nic

α4-eGFP β2-wt + 0.1 µM Nic

α4-eGFP β2-wt, No Nic

GalT-mcherry + 0.1 µM Nic

GalT-mcherry

α4-eGFP β2-DM

Merge

9


Changes in the brain during chronic exposure to nicotine

Specific Expression of a6 nAChRs in Midbrain DA Neurons

DA Neurons

GABA Neurons

WT

a6 L9′S

a4 L9′A

10


Changes in the brain during chronic exposure to nicotine

a4 Subunits are Required for Behavioral Hyperactivity

Observed in a6 L9’S Mice

11

Ryan Drenan, Sheri McKinney

Drenan et al., in preparation 2010


Changes in the brain during chronic exposure to nicotine

Conclusion: a4 Subunits are Critical to a6 nAChR Function In Vivo

12


Changes in the brain during chronic exposure to nicotine

2nd 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).


Changes in the brain during chronic exposure to nicotine

Reliable α6β2 expression in N2a cells

B

A

Vm = -60 mV, 0.3 mM ACh (0.1 s puff)

Vm = -60 mV

Vm = -90 mV

30

60

1.93 pA → 21.4 pS

1.44 pA → 24 pS

20

40

3 pA

Counts

15 ms

10

20

0

0

-2.0

-1.5

-1.0

-0.5

-3.0

-2.5

-2.0

-1.5

-1

C

Amplitude (pA)

Amplitude (pA)

D


Changes in the brain during chronic exposure to nicotine

Chronic nicotine exposure downregulates PM α6-meGFP β2 nAChRs

ER subtracted

No Nic

+ 0.1 µM Nic

ER subtracted


5 an accessory subunit

α5 an “accessory” subunit

  • 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


Two stoichiometries of 4 2 receptors

Two stoichiometries of α4β2 receptors

β2

β2

α4

α4

β2

α4

α4

β2

α4

β2

One stoichiometry of α4β2α5 receptors?

α4

β2

β2

α5

α4


Changes in the brain during chronic exposure to nicotine

HEK 293

1

3

4

2

500ng ea.

6

8

5

7

  • 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

  • α4β2 α5-IDT D/N C-Term GFP

  • α4β2 β3P379 YFP

9

10


Moving forward with 5

Moving Forward with α5. . .

  • 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


Changes in the brain during chronic exposure to nicotine

Lynx proteins modulate (inhibit) nicotinic receptor function 1-2Lynx proteins are GPI-linked variants of -neurotoxins (e.g.  -bungarotoxin) 1Removal of lynx using genetically engineered mice cause nicotinic receptor hypsersensitivity and enhanced learning and memory 3

  • Miwa et al., Neuron, 1999

  • Ibanez-Tallon et al., Neuron, 2002

  • 3. Miwa et al., Neuron, 2006

lynx btx


Changes in the brain during chronic exposure to nicotine

  •  -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.

 btx and AChBP


Changes in the brain during chronic exposure to nicotine

Questions:Do nAChR co-localize with lipid rafts? Labeling rafts by incubating transfected cells with labeled cholera toxin1) co-labeling: 4b2 receptors fused in-frame to gfp/cherry2) 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-b –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


Changes in the brain during chronic exposure to nicotine

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

23


Changes in the brain during chronic exposure to nicotine

Differential effects of nicotinic ligands on the assembly, ER exit and PM localization of nAChRs

Merge

No Drug

Sec24D-eGFP

+ 0.1 µM Nic, 48 h

4-mCherry + 2-eGFP

+ 0.1 µM Cyt, 48 h

10 µm

24


Changes in the brain during chronic exposure to nicotine

Differential effects of nicotinic ligands on the assembly, ER exit and PM localization of nAChRs

Merge

No Drug

Sec24D-eGFP

+ 0.1 µM Nic, 48 h

4-mCherry + 2-eGFP

10 µm

25


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