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Epigenetic Effects In Substance Abuse => Chromatin Therapeutics

Epigenetic Effects In Substance Abuse => Chromatin Therapeutics

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Epigenetic Effects InSubstance Abuse

=>

Chromatin Therapeutics

Alexander J Annala, PhD

Newton Clinical Research Group

UCLA ISAPI NPI&H

Motivation

Setting Priorities for Basic Brain & Behavioral Science at NIMH Final Report of the National Advisory Mental Health Council’s Workgroup on Basic Sciences – 5/2004 – Page 4

"This emerging field of epigenetics has received relatively little attention at NIH, as the tools for analysis of such

interaction have not yet been solidified. The Workgroup considers it time to invest more in developing the tools that

will allow intensive study in this area, given that most mental disorders appear to involve or result from such dynamic

processes over time.“

Speculative Discussion

While The Evidence Is Not Rock Solid – There Is Sufficient Information Available To Recommend Increased Attention

To Potential Epigenetic Effects In Substance Abuse

Speculative – Per Oxford English Dictionary 2nd Edition

Of the nature of, based upon, characterized by, speculation

or theory in contrast to practical or positive knowledge

Classical Genetics

DNA => RNA => PROTEIN

The Central Dogma

Human Genome Project

Describe Shared Human DNA Map

Search For DNA Mutations

Permanent A C T G Base Changes

Insertions, Deletions & Substitutions

=>

Disease & Pathological Conditions

DNA Mutations

Can Be Measured By Many Methods

Polymerase Chain Reaction (PCR)

DNA Gene Chip Analysis

DNA Mutations

Can Lead To

Ablation of Common Genes

or

Addition of Novel Genes

or

Changes In Protein Product Structure

DNA Mutations

Changes Are Generally Reflected

In Every Cell Throughout The Body

When Mutation Occurs In A Germ Cell

or

In One Specific Cell Line

When Mutation Occurs In A Somatic Cell

Differentiation & Development

Do Not Involve DNA Mutation

Do Involve Changes In Gene Expression

Changes Restricted To Specific Cell Lineage

Differentiation & Development

Pancreatic Islet Cells and Hepatocytes

Are Derived From A Common Progenitor

Cell Line – But Differ From Each Other

By Means Of Long Lasting Changes In

Silencing Or Expression Of Specific Genes

HNF-4 Expression => Hepatocyte-Like Cells

Changes In Gene Expression

Can Be Measured By Many Means

RNA Reverse Transcriptase PCR

RNA Gene Chip Analysis

Principal Epigenetic Phenomena

DNA Cytosine-5 Methylation

and / or

Histone H3 & H4 Tail Acetylation

Epigenetic Phenomena

Control Dynamic Changes In Gene Expression

Not Based on Permanent DNA Mutation

Epigenetic Phenomena

While Epigenetic Changes In Gene Expression

Are Not Permanent They Can Be Long Lasting

Routine Epigenetic Changes

DNA Methylation + Histone Tail Acetylation

Changes In Early Childhood

Silence Fetal Hemoglobin Gene Expression

Activate Adult Hemoglobin Gene Expression

These Changes Last An Entire Lifetime

Pathologic Epigenetic Changes

DNA Methylation + Histone Acetylation In

Fragile X Mental Retardation Silences FMR-1

These Changes Last An Entire Lifetime

Epigenetic Phenomena

DNA Methylation Changes

&

Histone Modifications

=>

Remodeling of Chromatin

DNA Methylation Changes

Cytosine-5 Methylation By

DNA Methyltransferase (DNMT)

cggcggcggcggcggcggcggc

=>

m m m m m m m m

cggcggcggcggcggcggcggc

DNA Methylation

Interferes With Formation of

Transcription Factor Complex

(DNA => RNA Initiation)

DNA Methylation

Interfers With Transcription Of

DNA Into RNA

By Retarding The Progress Of

DNA => RNA Transcriptase

Reducing Amount of Protein Product

(DNA => RNA Precession)

DNA Methyltransferase Inhibitors (DNMTi)

5-aza-cytidine

5-aza-2-deoxycytidine

MG98 [Methylgene Inc]

Zebularine [Eric Selker, U Oregon])

=> Orally Bioavailable Nontoxic

Zebularine (DNMTi)NIH / NIGMS FY 2005 Budget Page 12

Eric Selker, PhD demonstrated Zebularine PO reverses DNA methylation and reactivates a tumor suppressor gene in human bladder cancer injected into mice. Zebularine

reduced tumor size in the mice, making zebularine the first DNA methylation inhibitor to have such an effect in animals.

Zebularine is unique among the DNA methylation inhibitors that have been studied to date because it is chemically stable,

can be administered orally, and appears to be minimally toxic. Its only side effect seems to be a slight weight loss in

the mice given the chemical.

Zebularine (DNMTi)

J Natl Cancer Inst. 2003 Mar 5;95(5):399-409

Chromatin

The Combination Of

DNA Base Pairs ( A C T G ),

DNA Structure (Coils, Supercoils) And

DNA Packaging Materials

Chromatin

May Be Conceptually Visualized

As A Long Strand Of DNA (A C T G)

Which May Be Marked (C5-Methylated)

and / or

Which May Be Wound Around

A Long Series of Tiny Spools (Histones)

Chromatin

May Take One Of Two Primary Forms

Euchromatin (Active/Expressed)

or

Heterochromatin (Repressed/Silent)

Histone H3/H4 Tail Acetylation

Histone Tail Acetylation Is Controlled By

Dynamic Balance Between Enzyme Activities

Histone Acetyl Transferase (HAT)

and

Histone Deacetylase (HDAC)

Histone H3/H4 Tail Acetylation

Histones H3 and H4 May Be

Conceptually Visualized As A Long Series Of Tiny Spools Around Which Short

Segments Of One Long Strand Of

DNA May Be Wound

Histone H3/H4 Tail Acetylation

Histones H3 and H4 Have Tails

Which May Be Conceptually Visualized

As Four Short Protein Strands

Extending Away From

Each Histone Spool

Histone H3/H4 Tail Acetylation

H3/H4 Tail Acetylation

Stiffens Each Tail Increasing Space

Occupied By Each Histone Spool

H3/H4 Tail DeAcetylation

Relaxes Each Tail Compacting Space Occupied By Each Histone Spool

Chromatin

Increased Acetylation of H3/H4 Tails Forms Euchromatin

Stiffer Tails Make It Difficult For DNA To Be Tightly Compacted Leaving DNA Readily

Accessible To Initiation and Precession of DNA=>RNA Transcription Machinery

=>

Increased Gene Expression

Chromatin

Decreased Acetylation of H3/H4 Tails Forms Heterochromatin

Flexible Tails Make It Easy For DNA To Be Tightly Compacted Making DNA Less

Accessible To Initiation and Precession of DNA=>RNA Transcription Machinery

=>

Decreased Gene Expression

Chromatin

Increased Acetylation of H3/H4 Tails =>

Euchromatin =>

Increased Gene Expression

Decreased Acetylation of H3/H4 Tails => Heterochromatin =>

Decreased Gene Expression

Histone Deacetylase Inhibitors (HDACi)

Trichostatin A (TSA)

Valproic Acid (Depakote, Depakene)

Butyrate (ArgBu, NaBu, BA, 4-PBA)

SAHA,

CHR-2504 [Chroma Therapeutics], and

MGCD0103 [Methylgene Inc])

Sickle Cell Anemia

Symptoms Of Sickle Cell Anemia,

B-Thalassemia & Similar Conditions

May Be Relieved By Reactivation Of

Fetal Hemoglobin Expression

Using Histone Deacetylase Inhibitor

Arginine-Butyrate (HDACi)

Sickle Cell + HDACi

N Engl J Med. 1993 Jan 14;328(2):81-6

Fragile X Mental Retardation

FRAXA Is Usually Characterized By

DOUBLE LOCK

On FMR-1 Gene Expression Due To

DNA Methylation

And

Histone Deacetylation

Fragile X => DOUBLE LOCK

DNA Methylation + Histone Deacetylation => Heterochromatin Hum Mol Genet. 1999 Nov;8(12):2317-23

Fragile X Mental Retardation

FRAXA May Ultimately Be Treated By

Either HDACi Or DNMTi Alone

Or

By HDACi + DNMTi Combination

HDACi + DNMTi => Synergy

Fragile X + HDACi

Hum Mol Genet. 1999 Nov;8(12):2317-23

Fragile X + HDACi

Hum Mol Genet. 1999 Nov;8(12):2317-23

Fragile X + DNMTi

Hum Mol Genet. 1999 Nov;8(12):2317-23

Fragile X + HDACi +DNMTi

Synergism 0.2 or 1.0 uM 5azaDC + 5um BA or 4-PBA (Open = 5-azaDC Black=5azaDC +(BA or 4-PBA) Hum Mol Genet. 1999 Nov;8(12):2317-23

Histone Code Hypothesis

Histone H3 Methylated At Lysine 9

=>

Heterochromatin (Silent/Repressed)

Histone H3 Methylated At Lysine 4

=>

Euchromatin (Expressed/Active)

Histone Code Hypothesis

There Are Numerous Additional

Histone Modifications Leading

To Specific Activational Effects

However H3-K4 / H3-K9 Are

The Most Comprehensively Characterized

Modifications Identified To Date

Ethanol Addiction

SAMe Improves Performance of

Alcoholics On Psychometric Tests

Vittorio Coiro & Pier Paolo Vescovi

Controlled Study of Psychometric Performance In Abstinent Alcoholics:

Masked Comparison Of The Effects Of 15 Day Intravenous Treatments

With S-Adenosylmethionine Or Normal Saline

Current Therapeutic Research 58(9):575-586 (1997)

Ethanol Addiction

ETOH Demethylates NMDA-NR2B Promoter => Upregulating NR2B Expression

C R Marutha Ravindran & Maharaj K Ticku

Changes In Methylation Pattern Of NMDA Receptor NR2B Gene In

Cortical Neurons After Chronic Ethanol Treatment In Mice

Molecular Brain Research 121:19-17 (2004)

Ethanol Addiction

Pre-Conception Sire Exposure To EOTH =>

Degrades Mouse Pup Maze & Reflex Performance

Patricia A Jamerson, Michael J Wulser, Bruce F Kimler

Neurobehavioral Effects In Rat Pups Whose Sires Were Exposed To Alcohol

Developmental Brain Research 149:103-111 (2004)

Ethanol Addiction

Hepatocyte H3K9 Acetylated 8-10X By 5mM ETOH

Phil-Hoon Park, Rebecca Miller & Shivendra D Shukla

Acetylation Of Histone H3 At Lysine 9 By Ethanol In Rat Hepatocytes

Biochemical & Biophysical Research Communications 306:501-594 (2003)

Cocaine Addiction

cFos Promoter H4 Acetylation Increases After A Single Dose –

Not After Chronic Administration Of Cocaine.

This Is Consistent With Cocaine's Ability To Induce cFos Gene Expression Acutely But Not Chronically.

A. Kumar, K.H. Choi, N. Tsankova, E. Ruiz-Durantez, D.W. Self, E.J. NestlerSociety For Neuroscience Annual Meeting 2004 Poster # 692.4

Cocaine Addiction

BDNF & Cdk5 Promoter H3 Acetylation Increases

After Chronic Administration Of Cocaine

This Is Consistent With Cocaine’s Ability To Induce BDNF & Cdk5 Gene Expression Chronically But Not Acutely

BDNF & Cdk5 Promoter H3 Acetylation Was Higher In Cocaine

Self-Administering Rats Than In Chronically Injected Rats.

A. Kumar, K.H. Choi, N. Tsankova, E. Ruiz-Durantez, D.W. Self, E.J. NestlerSociety For Neuroscience Annual Meeting 2004 Poster # 692.4

Cocaine Addiction

TREAT AS CONFIDENTIAL TO COURSE MEMBERS

A Histone Deacetylase Inhibitor

. . . Potentiated Cocaine-Induced Locomotor Activity

Arvind Kumar, Eric Nestler, Personal Communication, 2005

Methamphetamine (MAP) Addiction

3 Hours After 4mg/kg MAP Dose In Mice

DNMT1 mRNA Decreases In Hippocampus

Yohtaro Numachi, Sumiko Yoshida, Motoyasu Yamashita, Ko Fujiyama,

Maki Naka, Hiroo Matsuka, Mitsumoto Sato & Ichiro Sora

Psychostimulant Alters Expression of Methyltransferase mRNA In Rat Brain

Annals New York Academy of Sciences 1025:102-109 (2004)

Methamphetamine (MAP) Addiction

3 Hours After 4mg/kg MAP Dose In Mice

Reelin mRNA Decreases 28%

In Frontal Cortex

Yohtaro Numachi, Sumiko Yoshida, Motoyasu Yamashita, Ko Fujiyama,

Maki Naka, Hiroo Matsuka, Mitsumoto Sato & Ichiro Sora

Psychostimulant Alters Expression of Methyltransferase mRNA In Rat Brain

Annals New York Academy of Sciences 1025:102-109 (2004)

Methamphetamine (MAP) Addiction

24 Hours After 4mg/kg MAP Dose In Mice

DNMT2 mRNA Decreases 27% To 39%

In Hippocampus Dentate Gyrus, CA1 & CA3

Yohtaro Numachi, Sumiko Yoshida, Motoyasu Yamashita, Ko Fujiyama,

Maki Naka, Hiroo Matsuka, Mitsumoto Sato & Ichiro Sora

Psychostimulant Alters Expression of Methyltransferase mRNA In Rat Brain

Annals New York Academy of Sciences 1025:102-109 (2004)

Methamphetamine (MAP) Addiction

MAP Can Alter DNA Methylation And Gene

Expression In Hippocampus & Frontal Cortex

Yohtaro Numachi, Sumiko Yoshida, Motoyasu Yamashita, Ko Fujiyama,

Maki Naka, Hiroo Matsuka, Mitsumoto Sato & Ichiro Sora

Psychostimulant Alters Expression of Methyltransferase mRNA In Rat Brain

Annals New York Academy of Sciences 1025:102-109 (2004)

Methamphetamine (MAP) Addiction

Decreased Reelin mRNA In Frontal Cortex Is Similar To Heterozygous Reeler Mice

May Be Related To Schizophrenia Like

Psychotic Symptoms of MAP Psychosis

Yohtaro Numachi, Sumiko Yoshida, Motoyasu Yamashita, Ko Fujiyama,

Maki Naka, Hiroo Matsuka, Mitsumoto Sato & Ichiro Sora

Psychostimulant Alters Expression of Methyltransferase mRNA In Rat Brain

Annals New York Academy of Sciences 1025:102-109 (2004)

Tremolizzo Schizphorenia Mice

Complementary Therapeutic Effects Obtained With

L-Methionine PO => DNA Methylation =>

Reelin/GAD67 Decrease => Long Delay PPI Deficit

Valproic Acid PO => Histone Acetylation =>

Reelin/GAD67 Normalized => PPI Remission

DNMTi/HDACi Pharmaceuticals

Chroma Therapeutics

Abingdon, Oxon, United Kingdom

http://www.chromatherapeutics.com/

Methylgene Inc

Montreal, Quebec, Canada

http://www.methylgene.com/

Conclusions

Epigenetic Phenomena May Form Substrates For Long Lasting Effects In Substance Abuse

Emerging Chromatin Therapeutic Agents

Offer Opportunities To Disrupt Neurological Adaptations Underlying Substance Abuse