epigenetic effects in substance abuse => chromatin therapeutics
TRANSCRIPT
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
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
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
DNA Methylation Changes
Cytosine-5 Methylation By
DNA Methyltransferase (DNMT)
cggcggcggcggcggcggcggc
=>
m m m m m m m m
cggcggcggcggcggcggcggc
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.
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)
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 +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