Identifying Genes that Impact Methamphetamine Use Disorder Tamara - - PowerPoint PPT Presentation

Identifying Genes that Impact Methamphetamine Use Disorder

Tamara J. Phillips, Ph.D.

VA Senior Research Career Scientist Director Scientific Director Professor of Behavioral Neuroscience

Methamphetamine (MA)

• Speed, ice, crystal
• Unpredictable and sometimes lethal
• Remains a significant drug threat
• 2017 – National Survey on Drug Use and Health (NSDUH): 1.6

million people in the U.S. reported using MA in the past year.

• Average age of users is 23 y.o.
• About 1 million had a MA use disorder
• Highest availability in the western and Midwestern U.S.

Pacific Northwest: Drug of Choice is MA

The Columbian Newspaper, Vancouver WA

Age-adjusted Rate of MA Overdose Deaths in 2017

SOURCE: NCHS National Vital Statistics System, Mortality files linked with death certificate lit

Adverse Health Effects

• Weight loss
• Severe dental problems
• Intense itching, leading to skin sores
• Anxiety, confusion, paranoia, hallucinations
• Memory loss
• Sleep problems
• Changes in brain structure and function
• Those using by injection have increased risk of contracting

infectious diseases

Individual Vulnerability

• Not all initial MA users go on to use regularly or to develop

MA addiction.

• Genetic factors may influence sensitivity to MA.
• Genetic factors may influence the amount of MA use.
• Selective breeding provides a tool for examining genetic

influences on behavior.

0 1 2 3 4 5 6 7

Selective Breeding for Voluntary Methamphetamine Intake

1st selected generation (60/line) 2nd selected generation (60/line) 3rd selected generation (60/line) Originating Population (120 mice) (a genetically heterogeneous sample)

Frequency:

10 20 10 20 10 20 10 20

MA Consumed (mg/kg): Selection diagram credited to Dr. John K. Belknap

Measurement of Voluntary Methamphetamine Drinking

Days 1 & 2 Days 3-6 Habituation 20 mg/L MA vs. Water Days 7-10 40 mg/L MA vs. Water = water tube = MA tube Days (4+6)/2 and (8+10)/2= mean intake (mg/kg) for each concentration

Day 3-10 daily timeline:

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Hours

water water

• -----------water & methamphetamine------------

Replicable Results

F2=Second filial cross of the D2 and B6 inbred strains MALDR=MA low drinking line; MAHDR=MA high drinking line

Cheryl Reed Jason Erk Harue Baba Jeanna Wheeler Zeni Shabani

Selection Generation

S0 S1 S2 S3 S4

MA Consumption (mg/kg/18 hrs; 40 mg MA/l)

2 4 6 8

F2 MALDR MAHDR F2-2 MALDR-2 MAHDR-2 F2-3 MALDR-3 MAHDR-3 F2-4 MALDR-4 MAHDR-4 F2-5 MALDR-5 MAHDR-5

MAHDR MALDR

Results for Several Phenotypes Compared in the MA Drinking Lines

Phenotypes for which MAHDR > MALDR:

MA drinking; 2-bc, 4-bc escalating concentration and lickometer MA blood levels AFTER MA drinking Operant ICV and operant oral MA SA MA-induced CPP, acute activation, sensitization and HYPERthermia

Phenotypes for which MAHDR < MALDR:

MA-induced CTA, CPA and HYPOthermia Spatial memory retention Morphine drinking and locomotor effects

Phenotypes for which MAHDR = MALDR

Quinine, saccharin and KCl drinking; 2-bc Basal locomotor activity and MA-induced locomotor stimulation MA clearance rate Novel object recognition, spatial memory learning and fear conditioning Ethanol-induced HYPOthermia COC-induced CPP, CTA and locomotor stimulation

Conditioned Taste Aversion (CTA)

Day 1-5 Day 6 Day 7, 9, 11, 13, and 15 Reduce access to water so that the mice will be thirsty and will learn to drink at a particular time

• f day.

Drink novel salty water. The purpose of this day is to introduce the novel taste and reduce neophobia. Drink salty water, then inject saline or MA. Thus, the effect of the injected solution is paired with the mostly novel taste. If the effect of MA “feels good (or doesn’t feel bad)” the mice will continue to drink the salty water the NEXT day. If it “feels bad” the mice will reduce the amount of salty water they drink the next day.

The MA Drinking Lines Differ in Sensitivity to MA- induced CTA

Wheeler et al. (2009) Genes Brain Behavior 8:758; Shabani et al. (2012) Neuropharmacology 62:1134

The MA Drinking Line Mice do NOT Differ in CTA Response to Cocaine

Gubner et al. (2013) Behav Brain Res 256:420-427

MA drinking Tube to Water Tube Ratio Impacts MA Intake

Shabani et al. (2016) Front Neurosci 10:493

Inspired by: Tordoff MG & Bachmanov AA (2003) Influence

• f the number of

alcohol and water bottles on murine alcohol intake. Alcohol Clin Exp Res 27:600.

Measuring Sensitivity to Drug Reward (and Aversion)

Place Conditioning set-up during testing

Hole floor Grid floor

Drug Absent Test: Are the cues that were previously paired with the drug preferred

• r avoided?

Drug Present Test: What is the impact on preference/avoidance when the animals are in the drug-treated state?

Image contributed by Dr. Chris Cunningham

MA-induced CPP in MAHDR Mice in the Drug-Free Test

Drug-Free CPP Test

***

MAHDR MALDR MAHDR: Spent ~60% of their time

• n MA-paired floor

MALDR: Spent ~50% of their time

• n the MA-paired floor

Shabani et al. (2011) Gene Brain Behav 10:625

CPA in MALDR Mice in the Presence of MA Treatment

*** ***

MA-Present CPP Test MAHDR MALDR MAHDR: Spent ~66% of their time on the 0.5 mg/ kg MA-paired floor. MALDR: Spent ~33% of their time on the drug- paired floor.

Shabani et al. (2011) Gene Brain Behav 10:625

Quantitative Trait Locus (QTL) Mapping

D2 alleles increase MA intake B6 alleles increase MA intake Taar1

Belknap et al. (2013) Mamm Genome 24:446-58

MA Drinking is Associated withTaar1 Genotype (Chr 10: 23.92 Mb)

Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184

= genotypes lacking TAAR1 receptor function John Harkness

Body Temperature Response to MA is Associated with Taar1 Genotype

Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184

Response is to 2 mg/kg MA. There were no differences after saline treatment

Sensitivity to MA-induced CTA is Associated with Taar1 Genotype

Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184.

Topology of Mouse Trace Amine-Associated Receptor 1

DBA/2J (D2) mice possess a unique non-synonymous SNP in Taar1 that is not present in 28 other strains of mice that have been genotyped. The mutation arose spontaneously. D2 is one of the progenitor strains of the MA drinking lines.

Figure adapted from Lindemann et al., 2005, Genomics 85:372

The mouse SNP encodes a proline to threonine mutation that predicts a change in receptor conformation. This is not a deletion mutation, but we have determined that the receptor expressed by the mutant allele (Taar1m1J) is nonfunctional.

TAAR1

The Mutant Allele Codes for a TAAR1 that is not Activated by MA in Transfected Cells

Confocal images

• f HEK293 cells

transfected with GFP-tagged B6-

• r D2-like Taar1.

GFP immunodetection indicates that both alleles express mTAAR1 in HEK293 cells. Human variants also have differential function.

Harkness et al. (2015) Neuropsychopharmacology 40:2175-2184

Trace Amine-Associated Receptor Genes

Eyun et al. (2016) PLoS One 11:e0151023

Trace Amine-Associated Receptor 1: TAAR1

• All mouse TAARs except TAAR1 are expressed in the main olfactory
• epithelium. TAAR1 is expressed in reward-related brain areas.
• TAAR1 is a cytosolic, stimulatory G-protein-coupled receptor that is

responsive to trace amines, such as β-phenylethylamine, octopamine, and tyramine.

• Methamphetamine is a TAAR1 agonist.
• TAAR1 activation triggers accumulation of intracellular cAMP, and appears to

modulate PKA and PKC signaling, and interfere with the β-arrestin2-dependent pathway via G protein-independent mechanisms.

• TAAR1 appears to exert control over monoamine neuronal firing and release;

unclear whether this is a direct or indirect effect. Greater dopamine in the absence of TAAR1 function has been reported.

Knock-in Recovery of TAAR1-associated MA Traits

Alex Stafford Cheryl Reed

Recovery of low MA intake No impact on fluid intake

Stafford, Reed et al. (2019) eLife 8:46472

Chronology for the Taar1m1J Allele

X

Reed et al. (2018) Front Pharmacol 8:993

Knock-in Recovery of TAAR1-associated MA Traits

Alex Stafford Cheryl Reed

Recovery of low MA intake No impact on fluid intake

Stafford, Reed et al. (2019) eLife 8:46472

Range of Consumption Values in S4 Generation of the Replicate 3 MADR Lines

MADR Line and Sex

MAHDR F MALDR F MAHDR M MALDR M

MA consumption (mg/kg/18 hrs; 40 mg MA/L)

2 4 6 8 10 12 14

Note that only those individual data points at locations outside of the boxes are shown

Phillips and colleagues, unpublished

Individual Variation and Patterns of MA Intake in MAHDR Mice

These data are for mice that had daily access to 3 bottles of 80 mg/L MA and 1 bottle of water, which results in higher levels of intake.

Shabani et al. (2016) Front Neurosci 10:493.

Why is There Modest Variability in MA Intake in MALDR, and Considerable Variability in MAHDR Mice?

• When TAAR1 function is absent, rewarding effects of MA can be
• experienced. - MAHDR exhibit MA reward and reinforcement as a group;

MALDR do not

• Did our selection not completely fix those genes that impact MA reward

in the MAHDR line?

• Are there modifiers in the genetic background that impact the Taar1m1J/

m1J effect on MA intake, resulting in lower levels of MA intake in some

MAHDR individuals?

• Are there epigenetic (environmental) effects that can modify the impact
• f the mutant allele?

MA-induced CPP in MAHDR Mice in the Drug-Free Test

Drug-Free CPP Test

***

MAHDR MALDR

NOT a TAAR1 effect, because TAAR1 is non-functional Mixture of TAAR1 and non-TAAR1 (e.g., dopamine) effects of MA?

MAHDR: Spent ~65% of their time

• n MA-paired floor

MALDR: Spent ~50% of their time

• n the MA-paired floor

Shabani (2011) Gene Brain Behav 10:625

The TAAR1 Agonist, RO-5256390, Induces Conditioned Place Aversion (CPA) in MALDR Mice

Drug-Free CPP Test Spent ~33% of their time on the drug-paired floor – clear avoidance. In the same procedure with doses of 0.5, 2 and 4 mg/kg, the MALDR mice were neutral. No preference and no avoidance.

Shabani, Phillips et. al. , unpublished

Extreme CPA When the TAAR1 Agonist is On-Board During the Preference Test in MALDR Mice

RO-Present CPP Test

Shabani, Phillips et. al. , unpublished

Spent ~15% of their time on the drug-paired floor – even clearer avoidance. In the same procedure with doses of 0.5, 2 and 4 mg/kg, the MALDR mice spent ~33% of their time on the drug-paired floor. Other effects of MA partially

• verride the TAAR1-specific,

aversive effects.

Heterogeneous Stock Mice

Brynn Voy, ORNL Mammalian Genome 2008

Inbreeding Outbreeding

Heterogeneous Stock – CC (HS-CC) are similar to the Diversity Outbred

Methamphetamine Intake in HS-CC Mice

40 mg/l MA MA consumed (mg/kg18 h)

0 1 2 3 4 5 6 7 8 9 10 11

Frequency

10 20 30 40 50 60

Total N = 57

Mean=0.6 mg/kg

Selection Results for HS-CC x D2 Cross

MAH = selected for high MA intake

• all Taar1m1J/m1J

MAL = selected for low MA intake - also all Taar1m1J/m1J MAC = Control line - all Taar1+/+

Searching for Genetic Modifiers in the Selected Lines

• Take brain samples from MA-naïve S4 offspring of the MAH, MAL and MAC

lines.

– When we did this in MAHDR vs. MALDR, glutamate-mediated synaptic plasticity was identified as having a key role in the outcome of selective breeding.

• Perform RNA-Seq analysis on this tissue, first comparing MAH to MAL – gene

expression/network differences will point to potential modifiers of the Taar1m1J/

m1J genotype effect.

• The genes/networks identified may also be important for sensitivity to the

rewarding effects of MA, and these lines will be tested for MA-induced CPP and

• perant self-administration.
• Our initial concentration will be on the medial shell of the nucleus accumbens,

but other tissues will be retained.

Next Steps

• Evaluate individual genes that are associated with variation in MA intake for

differential expression, differential variability and differential wiring.

• Identify network “hubs” – hubs are highly interconnected/coexpressed genes.
• Hubs that are significantly rewired in coordination with variation in MA intake,

are key targets for manipulation.

• Manipulate hubs to determine if they strongly regulate MA intake.
• Manipulations could include the use of pharmacological agents, mutant mice,

siRNA for gene knockdown, etc.

• This may lead to new treatment targets for clinical translation; they may be

downstream effects of TAAR1 activation.

An Alternative Signaling Pathway for TAAR1: RhoA

Underhill et al. (2019) Molecular Psychiatry [epub]

• MA enters the cell through the

DAT at the plasma membrane.

• Once inside, it binds to G13-

coupled TAAR1 receptors.

• This stimulates RhoA activation

near the ER.

• RhoA mediates endocytosis of

the DAT and the glutamate transporter, EAAT3.

• MA also stimulates GS-coupled

TAAR1 receptors that propagate PKA signaling.

• Downstream PKA activation

leads to phosphorylation of RhoA and stops transporter internalization.

Acknowledgements

• John Belknap
• Chris Cunningham
• Bob Hitzemann
• Aaron Janowsky
• Shannon McWeeney
• Kim Neve
• Cheryl Reed
• Kristine Wiren

Technical Assistance

• Sue Burkhart-Kasch
• Harue Baba
• Jason Erk
• Joel Hashimoto
• Na Li
• Carrie McKinnon
• Zhen (Andy) Zhu
• Emily Eastwood
• Fred Franken
• John Harkness
• John Mootz
• Angela Scibelli
• Zeni Shabani
• Xiao Shi
• Alex Stafford
• Jeanna Wheeler

Funding

• Dept. of Veterans Affairs

NIH/NIDA NIH/NIAAA

Collaborators Postdocs and Students