Epigenetic Effects In Substance Abuse => Chromatin Therapeutics

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Epigenetic Effects InSubstance
AbusegtChromatin Therapeutics
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Alexander J Annala, PhDNewton Clinical Research
GroupUCLA ISAPI NPIH
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Motivation

• Setting Priorities for Basic Brain Behavioral
  Science at NIMH Final Report of the National
  Advisory Mental Health Councils 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.

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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

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Classical Genetics

• DNA gt RNA gt PROTEIN
• The Central Dogma

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Human Genome Project

• Describe Shared Human DNA Map
• Search For DNA Mutations
• Permanent A C T G Base Changes
• Insertions, Deletions Substitutions
• gt
• Disease Pathological Conditions

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DNA Mutations

• Can Be Measured By Many Methods
• Polymerase Chain Reaction (PCR)
• DNA Gene Chip Analysis

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DNA Mutations

• Can Lead To
• Ablation of Common Genes
• or
• Addition of Novel Genes
• or
• Changes In Protein Product Structure

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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

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Differentiation Development

• Do Not Involve DNA Mutation
• Do Involve Changes In Gene Expression
• Changes Restricted To Specific Cell Lineage

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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 gt Hepatocyte-Like Cells

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Changes In Gene Expression

• Can Be Measured By Many Means
• RNA Reverse Transcriptase PCR
• RNA Gene Chip Analysis

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Principal Epigenetic Phenomena

• DNA Cytosine-5 Methylation
• and / or
• Histone H3 H4 Tail Acetylation

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Epigenetic Phenomena

• Control Dynamic Changes In Gene Expression
• Not Based on Permanent DNA Mutation

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Epigenetic Phenomena

• While Epigenetic Changes In Gene Expression
• Are Not Permanent They Can Be Long Lasting

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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

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Pathologic Epigenetic Changes

• DNA Methylation Histone Acetylation In
• Fragile X Mental Retardation Silences FMR-1
• These Changes Last An Entire Lifetime

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Epigenetic Phenomena

• DNA Methylation Changes
• Histone Modifications
• gt
• Remodeling of Chromatin

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DNA Methylation Changes

• Cytosine-5 Methylation By
• DNA Methyltransferase (DNMT)
• cggcggcggcggcggcggcggc
• gt
• m m m m m m m m
• cggcggcggcggcggcggcggc

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DNA Methylation

• Interferes With Formation of
• Transcription Factor Complex
• (DNA gt RNA Initiation)

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DNA Methylation

• Interfers With Transcription Of
• DNA Into RNA
• By Retarding The Progress Of
• DNA gt RNA Transcriptase
• Reducing Amount of Protein Product
• (DNA gt RNA Precession)

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DNA Methyltransferase Inhibitors (DNMTi)

• 5-aza-cytidine
• 5-aza-2-deoxycytidine
• MG98 Methylgene Inc
• Zebularine Eric Selker, U Oregon)
• gt Orally Bioavailable Nontoxic

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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.

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Zebularine (DNMTi)

• J Natl Cancer Inst. 2003 Mar 595(5)399-409

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Chromatin

• The Combination Of
• DNA Base Pairs ( A C T G ),
• DNA Structure (Coils, Supercoils) And
• DNA Packaging Materials

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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)

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Chromatin

• May Take One Of Two Primary Forms
• Euchromatin (Active/Expressed)
• or
• Heterochromatin (Repressed/Silent)

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Histone H3/H4 Tail Acetylation

• Histone Tail Acetylation Is Controlled By
• Dynamic Balance Between Enzyme Activities
• Histone Acetyl Transferase (HAT)
• and
• Histone Deacetylase (HDAC)

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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

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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

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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

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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 DNAgtRNA
  Transcription Machinery
• gt
• Increased Gene Expression

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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 DNAgtRNA
  Transcription Machinery
• gt
• Decreased Gene Expression

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Chromatin

• Increased Acetylation of H3/H4 Tails gt
• Euchromatin gt
• Increased Gene Expression
• Decreased Acetylation of H3/H4 Tails gt
  Heterochromatin gt
• Decreased Gene Expression

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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)

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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)

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Sickle Cell HDACi
N Engl J Med. 1993 Jan 14328(2)81-6
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Fragile X Mental Retardation

• FRAXA Is Usually Characterized By
• DOUBLE LOCK
• On FMR-1 Gene Expression Due To
• DNA Methylation
• And
• Histone Deacetylation

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Fragile X gt DOUBLE LOCK

• DNA Methylation Histone Deacetylation gt
  Heterochromatin Hum Mol Genet. 1999
  Nov8(12)2317-23

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Fragile X Mental Retardation

• FRAXA May Ultimately Be Treated By
• Either HDACi Or DNMTi Alone
• Or
• By HDACi DNMTi Combination
• HDACi DNMTi gt Synergy

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Fragile X HDACi

• Hum Mol Genet. 1999 Nov8(12)2317-23

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Fragile X HDACi

• Hum Mol Genet. 1999 Nov8(12)2317-23

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Fragile X DNMTi

• Hum Mol Genet. 1999 Nov8(12)2317-23

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Fragile X HDACi DNMTi

• Synergism 0.2 or 1.0 uM 5azaDC 5um BA or 4-PBA
  (Open 5-azaDC Black5azaDC (BA or 4-PBA) Hum
  Mol Genet. 1999 Nov8(12)2317-23

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Histone Code Hypothesis

• Histone H3 Methylated At Lysine 9
• gt
• Heterochromatin (Silent/Repressed)
• Histone H3 Methylated At Lysine 4
• gt
• Euchromatin (Expressed/Active)

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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

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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)

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Ethanol Addiction

• ETOH Demethylates NMDA-NR2B Promoter gt
  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 12119-17 (2004)

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Ethanol Addiction

• Pre-Conception Sire Exposure To EOTH gt
• 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 149103-111 (2004)

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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
  306501-594 (2003)

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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. Nestler
• Society For Neuroscience Annual Meeting 2004
  Poster 692.4

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Cocaine Addiction

• BDNF Cdk5 Promoter H3 Acetylation Increases
• After Chronic Administration Of Cocaine
• This Is Consistent With Cocaines 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. Nestler
• Society For Neuroscience Annual Meeting 2004
  Poster 692.4

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Cocaine Addiction

• TREAT AS CONFIDENTIAL TO COURSE MEMBERS
• A Histone Deacetylase Inhibitor
• . . . Potentiated Cocaine-Induced Locomotor
  Activity
• Arvind Kumar, Eric Nestler, Personal
  Communication, 2005

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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 1025102-109
  (2004)

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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 1025102-109
  (2004)

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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 1025102-109
  (2004)

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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 1025102-109
  (2004)

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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 1025102-109
  (2004)

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Tremolizzo Schizphorenia Mice

• Complementary Therapeutic Effects Obtained With
• L-Methionine PO gt DNA Methylation gt
• Reelin/GAD67 Decrease gt Long Delay PPI Deficit
• Valproic Acid PO gt Histone Acetylation gt
• Reelin/GAD67 Normalized gt PPI Remission

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DNMTi/HDACi Pharmaceuticals

• Chroma Therapeutics
• Abingdon, Oxon, United Kingdom
• http//www.chromatherapeutics.com/
• Methylgene Inc
• Montreal, Quebec, Canada
• http//www.methylgene.com/

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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

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