Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism
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Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism
Richard Deth, PhD Northeastern University Boston, MA
Overview
- Oxidation and Evolution - Regulation of Redox Status - Brain-specific Redox Features - Methionine synthase in human cortex - across the lifespan - in autism - Selenoproteins and mercury toxicity
rli est lif e ap pea rs t o h ave aris en at hydr othe rmal v en ts e mit ting dr oge n sulfi de and o ther ga ses at h igh t emp erat ure and pr essur
H2S H2O
Primordial Synthesis of Cysteine From Volcanic Gases
Methane Hydrogen sulfide Ammonia Carbon dioxide CH3 H2S NH3 CO2
NH2CHCOOH CH2 SH
Cysteine
Cysteine can function as an antioxidant
Two Antioxidant Reducing Equivalents
NH2CHCOOH CH2 SH
NH2CHCOOH
+
CH2 SH
NH2CHCOOH CH2 S + 2 H+ S CH2 NH2CHCOOH Cysteine Disulfide
Two Cysteines
Evolution = Adaptation to threat of oxidation O2 O2 Genetic Mutation
O2 O2
Novel Antioxidant Adaptation
=
Adaptive features of sulfur metabolism
Evolution = Metabolic Adaptations to an Oxygen Environment
Figure from Paul G. Falkowski Science 311 1724 (2006)
EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO ENVIRONMENTAL THREATS
The ability to control oxidation is at the core of evolution Each addition is strengthened because it builds on the solid core already in place.
New capabilities are added in the context of the particular environment in which they are useful and offer a selective advantage. Recently added capabilities are the most vulnerable to loss when and if there is a significant changes in the environment. Humans cognitive abilities are particularly vulnerable.
N LA GU AG E
SOCI
AL S
KILL
S
Oxidative Metabolism
Oxygen Radicals
Oxygen Radicals Genetic Risk Factors Redox Buffer Capacity
Redox Buffer Capacity [Glutathione]
OXIDATIVE STRESS
Heavy Metals + Xenobiotics
NORMAL REDOX BALANCE
Methylation Neuronal Synchronization
Neuronal Degeneration
Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell
Cysteine GSSG GSH
Glutathione γ-Glutamylcysteine Synthesis Cysteine Cystathionine
Adenosine
HCY
Methionine Synthase
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Dietary protein
Cognitive Status Catecholamine Methylation
Nitric Oxide Synthesis
Arginine Methylation
Gene Expression
REDOX STATUS: GSH GSSH
Methylation Status: SAM SAH
~ 200 Methylation Reactions
DNA/Histone Methylation Serotonin Methylation
Creatine Synthesis Energy Status
Phospholipid Methylation
Melatonin Membrane Properties
Sleep
During oxidative stress methionine synthase is turned off, allowing more homocysteine to flow toward GSH synthesis, while methylation activity is decreased
Cysteine GSSG GSH
Glutathione γ-Glutamylcysteine Synthesis Cysteine Cystathionine
OXIDATIVE STRESS
Adenosine
HCY
Methionine Synthase
SAH
MethylTHF
(-)
THF MET
ATP
>150 Methylati on
PP+Pi
SAM Reactons
Dietary protein
Inflammation is a metabolic state of oxidative stress, normally occurring in response to environmental challenges
Infection, allergy, trauma, chronic illness
Inflammatory State
- Survival mode - Loss of normal function
Recovery:
Adaptive responses to oxidative stress
- Impaired methylation
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Aging is associated with increased oxidative stress, as adaptive responses fail to restore normal redox status
↑ Inflammatory Diseases: - Alzheimer’s disease - Parkinson’s disease - Diabetes - Heart Failure
Aging
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Exposure to persistent environmental toxins promotes oxidative stress and impairs the ability to recover
Heavy Metal and Xenobiotic Exposure
Inflammatory State
- Survival mode - Loss of normal function - Impaired methylation - Autism??
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Autism is associated with oxidative stress and impaired methylation
28%↓
36%↓ 38%↓
he Brain Compartment (CSF) has low Thiol leve and maintains an Oxidative Stress environmen trocytes provide Cysteine to Neurons for surv BRAIN Neurons
[GSH] = 0.21mM [CYS] [CYS] [CysGly] [GSH] Blood-Brain Barrier
BLOOD
Astrocytes
[GSH] = 0.91mM [GSH] = 8μM [CYS] =200μM
[GSH] = 1 μM [CYS] = 2 μM
CSF
Neurons obtain cysteine from GSH released by Glial cells, via a growth factor-controlled transporter (EAAT3)
Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH γ-Glutamylcysteine Cysteine Cystathionine
Adenosine
PI3-kinase
HCY
Methionine Synthase
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Transsulfuration of homocysteine (HCY) to cysteine is restricted in human neuronal cells, increasing importance of cysteine uptake Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY
Methionine Synthase
PI3-kinase
Cysteine
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Methionine synthase in human neuronal cells requires methylB12 (MeCbl), whose synthesis is glutathione-dependent Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH GSCbl SAM MeCb l OHCbl γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY
Methionine Synthase
PI3-kinase
Cysteine H2S
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Levels of cystathionine are markedly higher in human cortex than in other species
Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.
In neurons, D4 dopamine receptors carry out phospholipid methylation, which requires methionine synthase to supply methyl groups Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH GSCbl SAM MeCb l OHCbl γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine Adenosine Cysteine
PI3-kinase
D4SAH
Phospholip id Methylatio n
D4HCY
HCY
SAH
MethylTH F THF
Methionine Synthase
MethylTHF
D4SAM
PP+Pi
D4MET
ATP
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Dopamine
DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION DOPAMINE
CH3
Methionine Synthase
Methylfolate
25
2 or 4-repeats
7-repeats
Methionine Synthase
Structure and function Brain levels Across the lifespan In autism
Methionine synthase has five domains + cobalamin (Vitamin B12) Domains alternate interacting with cobalamin during turnover
HCY Domain SAM Domain
3
2 1
Cobalamin (vitamin B12)
Cobalamin Domain
Cap Domain
5-Methyl THF Domain
3'
HCY
FOL
CAP
COB
SAM
5'
187 bp
197 bp
419 bp
Exon 19
188 bp
20
21
22
23
24
122 bp
25
Decrease of Cob domain mRNA with increasing age, 40 subjects
MS Cob mRNA (arbitrary units) 600 500 400 300 200 100 0 0 10 20 30 40 50 60 70 80 90 100 Age (years) T1/2fast = 3.4 years T1/2slow = 29.4 years R2 = .91
Decrease of Cap domain with increasing age, 40 subjects
MS Cap mRNA (arbitrary units) 700 600 500 400 300 200 100 0 0 10 20 30 40 50 60 Age (years) 70 80 90 T1/2fast = 2.2 years T1/2slow = 20 years R2 = .94
CAP Domain is present in MS mRNA from 24 y.o. subject
HCY
FOL
CAP
COB
SAM
CAP Domain is absent from methionine
synthase mRNA in elderly human cortex 80 year old subject
HCY FOL CAP COB SAM
Age-dependent decrease in the ratio of Cap to Cobalamin mRNA
80 year old subject
MS mRNA Cap/Cob Ratio
1.5
HCY
FOL
1.0
CAP
COB
SAM
0.5
0.0
0 er 2 U nd ve O r6 0
Alternative Splicing of MS Pre-mRNA leads to age-dependent exon skipping
Cap Domain Exons 19-21 Cap Domain Present
HCY
FOL
COB
SAM Cap Domain Absent
Site of alternative splicing by mRNA-specific adenosine deaminase
Pre-mRNA
mRNA
Exons 16-18 are deleted in fetal human brain
MS exists as two lower MW bands in SH-SY5Y cells Normal full size MW = 140 kDa
125 kDa Exons 16-18 are absent 110 kDa Exons 16-20 are absent
180 115 84
Methionine synthase activity can be regulated via multiple levels of control in response to oxidative stress
DNA
Transcription
Pre-mRNA
(introns + exons) Splicing
RNA
(exons only) Translation B12 Cofactor
Protein
mRNA for methionine synthase is 2-3 fold lower in cortex of autistic subjects as compared to age-matched controls
Age-dependent trend of methionine synthase CAP domain mRNA is absent in autism
MS Cap levels (arbitrary units)
400 300 200 100 0
T1/2 = 2.7 yrs r2 = 0.94
Controls Autistic
0
10
20
30
Age (years)
Paired comparisons of CAP domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)
400
Methionine Synthase CAP domain mRNA (arbitrary units)
1-5 yrs 6-10 yrs 11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs
300 200 100 0
Control
Autistic
Paired comparisons of Cob domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)
400
Methionine Synthase COB domain mRNA (arbitrary units)
300 200 100 0
1-5 yrs 6-10 yrs 11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs
Control
Autistic
Age-dependent changes in Cap and Cobalamin mRNA in Control vs. Autism
80 year old subject
HCY
5/4 FOL
CAP
11/9COB A
30/30 SAM
C
{ { {
A
C
{ { {
C
{ { {
A
Selenoproteins, mercury and redox status
Glucose is the major source of reducing power for maintaining reduced glutathione
NADP+
Glucose
NADPH Thioredoxin Reductase 6-P-gluconolactone Thioredoxin
Glucose-6-P
G6PD
GSH status
Thioredoxin reductase is a selenoprotein
NADP+
Glucose
NADPH Thioredoxin Reductase 6-P-gluconolactone Thioredoxin
Glucose-6-P
G6PD
CpG CpG CpG
G6PD gene (on)
GSH status Methionine Synthase Activity SAM SAH
DNA Demethylase
G6PD gene (off)
DNA Methyltransferase
CpG CpG CpG CH3 CH3 CH3
Hg
SULFUR AND SELENIUM AMINO ACIDS
H H3 N C CH 2 SH CYSTEINE Binding Constant = 1039 COO H3 N
H C CH 2 Se SELENOCYSTEINE Binding Constant = 1045 COO
Hg2+
(million-fold higher affinity)
From Dr. Nicholas Ralston Univ. of North Dakota
Mercury gradually migrates to highest affinity targets (i.e. selenoproteins)
Selenoproteins Thioredoxin fold proteins (dual stable thiols) Protein thiols (mono thiol sites) Thiol metabolites (GSH, cysteine)
Hg2+
Highest levels of GSH are in selenium-rich ependymal cells which are stem cells for astrocytes and neurons
Selenoprotein P Is high in Ependymal cells
Astrocyte [GSH] = 0.91 mM Neuron [GSH] = 0.21 mM
Ependymal [GSH] = 2.73 mM
Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006 Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007
Prevailing redox conditions determine the proportion of neurons vs. astrocytes which develop from neuronal stem cells
Pluripotent Stem Cells (Ependymal Cells) Oxidized State Pluripotent Stem Cells (Ependymal Cells) Normal State Pluripotent Stem Cells (Ependymal Cells) Reduced State
Less Neurons
More Astrocytes
Neurons
Astrocytes
More Neurons
Less Astrocytes
Neuronal cells
In human neuronal cells thimerosal partially inhibits the selenoprotein thioredoxin reductase with high potency, but inhibits thioredoxin with only low potency
Thioredoxin reductase
0.03
0.010
Thioredoxin
Enzyme Activity
Enzyme Activity
0.02
0.005
0.01
0.000 0
0.00
-12 -11 -10 -9 -8 -7 -6 -5 -4
0
-12 -11 -10
-9
-8
-7
-6
-5
-4
[Thimerosal] M
[Thimerosal] M
55
Thimerosal-induced reduction of GSH levels in SH-SY5Y human neuroblastoma cells
900 800 700 600 500 400 300 200 100 0
[GSH] (nmol / mg protein)
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Thimerosal] M
56
a
120
b
120 100 80 60 40 20 0 0 -11 -10 -9 -8 -7 -6 -5
MS activity pmol/min/mg protein
MS activity pmol/min/mg protein
Hydroxo-B12 Methyl-B12
100 80 60 40 20 0 0 -11 -10 -9 -8
Hydroxo-B12 Methyl-B12
-7
-6
-5
c
140
Log [Lead ] M
Hydroxo-B12 Methyl-B12
d
120
Log [Arsenic] M
Hydroxo-B12 Methyl-B12
MS activity pmol/min/mg protein
120 100 80 60 40 20 0 0 -12 -11 -10 -9
MS activity pmol/min/mg protein
100 80 60 40 20 0 0 -12 -11 -10 -9
-8
-7
-6
-5
-8
-7
-6
-5
e
100
Log [Aluminum] M
Hydroxo-B12 Methyl-B12
f
1750
Log [Mercury] M
Control Le ad Arse nic Aluminum M ercury Thime rosal
MS activity pmol/min/mg protein
[GSH] nmole/mg protein
80 60 40 20 0
1500 1250 1000 750 500 250 0
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Thimerosal] M
Genetic and Environmental Factors Can Combine to Cause Autism
Genetic Risk Factors Environmental Exposures
PON1, GSTM1
Impaired Sulfur Metabolism
Neuroinflammation
Oxidative Stress
MTRR, MTHFR, ADSL RFC, TCN2
Methionine Synthase Activity ↓
COMT, ATP10C, ADA MeCP2, ADA
↓ Receptor Phospholipid Methylation D4
MET, NLGN3/4 FMR-1, RELN
↓DNA Methylation ∆ Gene Expression Developmental Delay
↓ Neuronal Synchronization ↓ Attention and cognition
AUTISM
58
“… and a child shall lead them.”
Disorders sharing metabolic features with autism:
Attention-deficit hyperactivity disorder Alzheimer’s disease Schizophrenia Parkinson’s disease Chronic fatigue syndrome Amyotrophic lateral sclerosis Multiple sclerosis Type 2 diabetes Obesity
Thanks for your Research Support!! Autism Research Institute SafeMinds Cure Autism Now
Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism
Richard Deth, PhD Northeastern University Boston, MA
Overview
- Oxidation and Evolution - Regulation of Redox Status - Brain-specific Redox Features - Methionine synthase in human cortex - across the lifespan - in autism - Selenoproteins and mercury toxicity
rli est lif e ap pea rs t o h ave aris en at hydr othe rmal v en ts e mit ting dr oge n sulfi de and o ther ga ses at h igh t emp erat ure and pr essur
H2S H2O
Primordial Synthesis of Cysteine From Volcanic Gases
Methane Hydrogen sulfide Ammonia Carbon dioxide CH3 H2S NH3 CO2
NH2CHCOOH CH2 SH
Cysteine
Cysteine can function as an antioxidant
Two Antioxidant Reducing Equivalents
NH2CHCOOH CH2 SH
NH2CHCOOH
+
CH2 SH
NH2CHCOOH CH2 S + 2 H+ S CH2 NH2CHCOOH Cysteine Disulfide
Two Cysteines
Evolution = Adaptation to threat of oxidation O2 O2 Genetic Mutation
O2 O2
Novel Antioxidant Adaptation
=
Adaptive features of sulfur metabolism
Evolution = Metabolic Adaptations to an Oxygen Environment
Figure from Paul G. Falkowski Science 311 1724 (2006)
EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO ENVIRONMENTAL THREATS
The ability to control oxidation is at the core of evolution Each addition is strengthened because it builds on the solid core already in place.
New capabilities are added in the context of the particular environment in which they are useful and offer a selective advantage. Recently added capabilities are the most vulnerable to loss when and if there is a significant changes in the environment. Humans cognitive abilities are particularly vulnerable.
N LA GU AG E
SOCI
AL S
KILL
S
Oxidative Metabolism
Oxygen Radicals
Oxygen Radicals Genetic Risk Factors Redox Buffer Capacity
Redox Buffer Capacity [Glutathione]
OXIDATIVE STRESS
Heavy Metals + Xenobiotics
NORMAL REDOX BALANCE
Methylation Neuronal Synchronization
Neuronal Degeneration
Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell
Cysteine GSSG GSH
Glutathione γ-Glutamylcysteine Synthesis Cysteine Cystathionine
Adenosine
HCY
Methionine Synthase
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Dietary protein
Cognitive Status Catecholamine Methylation
Nitric Oxide Synthesis
Arginine Methylation
Gene Expression
REDOX STATUS: GSH GSSH
Methylation Status: SAM SAH
~ 200 Methylation Reactions
DNA/Histone Methylation Serotonin Methylation
Creatine Synthesis Energy Status
Phospholipid Methylation
Melatonin Membrane Properties
Sleep
During oxidative stress methionine synthase is turned off, allowing more homocysteine to flow toward GSH synthesis, while methylation activity is decreased
Cysteine GSSG GSH
Glutathione γ-Glutamylcysteine Synthesis Cysteine Cystathionine
OXIDATIVE STRESS
Adenosine
HCY
Methionine Synthase
SAH
MethylTHF
(-)
THF MET
ATP
>150 Methylati on
PP+Pi
SAM Reactons
Dietary protein
Inflammation is a metabolic state of oxidative stress, normally occurring in response to environmental challenges
Infection, allergy, trauma, chronic illness
Inflammatory State
- Survival mode - Loss of normal function
Recovery:
Adaptive responses to oxidative stress
- Impaired methylation
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Aging is associated with increased oxidative stress, as adaptive responses fail to restore normal redox status
↑ Inflammatory Diseases: - Alzheimer’s disease - Parkinson’s disease - Diabetes - Heart Failure
Aging
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Exposure to persistent environmental toxins promotes oxidative stress and impairs the ability to recover
Heavy Metal and Xenobiotic Exposure
Inflammatory State
- Survival mode - Loss of normal function - Impaired methylation - Autism??
GSH GSSG
= 30
GSH GSSG
= 10
Normal Redox Setpoint
Oxidative Stress
Autism is associated with oxidative stress and impaired methylation
28%↓
36%↓ 38%↓
he Brain Compartment (CSF) has low Thiol leve and maintains an Oxidative Stress environmen trocytes provide Cysteine to Neurons for surv BRAIN Neurons
[GSH] = 0.21mM [CYS] [CYS] [CysGly] [GSH] Blood-Brain Barrier
BLOOD
Astrocytes
[GSH] = 0.91mM [GSH] = 8μM [CYS] =200μM
[GSH] = 1 μM [CYS] = 2 μM
CSF
Neurons obtain cysteine from GSH released by Glial cells, via a growth factor-controlled transporter (EAAT3)
Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH γ-Glutamylcysteine Cysteine Cystathionine
Adenosine
PI3-kinase
HCY
Methionine Synthase
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Transsulfuration of homocysteine (HCY) to cysteine is restricted in human neuronal cells, increasing importance of cysteine uptake Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY
Methionine Synthase
PI3-kinase
Cysteine
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Methionine synthase in human neuronal cells requires methylB12 (MeCbl), whose synthesis is glutathione-dependent Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH GSCbl SAM MeCb l OHCbl γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY
Methionine Synthase
PI3-kinase
Cysteine H2S
SAH
MethylTHF
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Levels of cystathionine are markedly higher in human cortex than in other species
Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.
In neurons, D4 dopamine receptors carry out phospholipid methylation, which requires methionine synthase to supply methyl groups Growth Factors Cysteine Cysteinylglycine
(+)
GSH
Healthy Glial Cells (Astrocytes)
EAAT3 GSSG GSH GSCbl SAM MeCb l OHCbl γ-Glutamylcysteine PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine Adenosine Cysteine
PI3-kinase
D4SAH
Phospholip id Methylatio n
D4HCY
HCY
SAH
MethylTH F THF
Methionine Synthase
MethylTHF
D4SAM
PP+Pi
D4MET
ATP
THF MET
ATP
PP+Pi
>150 Methylati on SAM Reactons
(-)
Dopamine
DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION DOPAMINE
CH3
Methionine Synthase
Methylfolate
25
2 or 4-repeats
7-repeats
Methionine Synthase
Structure and function Brain levels Across the lifespan In autism
Methionine synthase has five domains + cobalamin (Vitamin B12) Domains alternate interacting with cobalamin during turnover
HCY Domain SAM Domain
3
2 1
Cobalamin (vitamin B12)
Cobalamin Domain
Cap Domain
5-Methyl THF Domain
3'
HCY
FOL
CAP
COB
SAM
5'
187 bp
197 bp
419 bp
Exon 19
188 bp
20
21
22
23
24
122 bp
25
Decrease of Cob domain mRNA with increasing age, 40 subjects
MS Cob mRNA (arbitrary units) 600 500 400 300 200 100 0 0 10 20 30 40 50 60 70 80 90 100 Age (years) T1/2fast = 3.4 years T1/2slow = 29.4 years R2 = .91
Decrease of Cap domain with increasing age, 40 subjects
MS Cap mRNA (arbitrary units) 700 600 500 400 300 200 100 0 0 10 20 30 40 50 60 Age (years) 70 80 90 T1/2fast = 2.2 years T1/2slow = 20 years R2 = .94
CAP Domain is present in MS mRNA from 24 y.o. subject
HCY
FOL
CAP
COB
SAM
CAP Domain is absent from methionine
synthase mRNA in elderly human cortex 80 year old subject
HCY FOL CAP COB SAM
Age-dependent decrease in the ratio of Cap to Cobalamin mRNA
80 year old subject
MS mRNA Cap/Cob Ratio
1.5
HCY
FOL
1.0
CAP
COB
SAM
0.5
0.0
0 er 2 U nd ve O r6 0
Alternative Splicing of MS Pre-mRNA leads to age-dependent exon skipping
Cap Domain Exons 19-21 Cap Domain Present
HCY
FOL
COB
SAM Cap Domain Absent
Site of alternative splicing by mRNA-specific adenosine deaminase
Pre-mRNA
mRNA
Exons 16-18 are deleted in fetal human brain
MS exists as two lower MW bands in SH-SY5Y cells Normal full size MW = 140 kDa
125 kDa Exons 16-18 are absent 110 kDa Exons 16-20 are absent
180 115 84
Methionine synthase activity can be regulated via multiple levels of control in response to oxidative stress
DNA
Transcription
Pre-mRNA
(introns + exons) Splicing
RNA
(exons only) Translation B12 Cofactor
Protein
mRNA for methionine synthase is 2-3 fold lower in cortex of autistic subjects as compared to age-matched controls
Age-dependent trend of methionine synthase CAP domain mRNA is absent in autism
MS Cap levels (arbitrary units)
400 300 200 100 0
T1/2 = 2.7 yrs r2 = 0.94
Controls Autistic
0
10
20
30
Age (years)
Paired comparisons of CAP domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)
400
Methionine Synthase CAP domain mRNA (arbitrary units)
1-5 yrs 6-10 yrs 11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs
300 200 100 0
Control
Autistic
Paired comparisons of Cob domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)
400
Methionine Synthase COB domain mRNA (arbitrary units)
300 200 100 0
1-5 yrs 6-10 yrs 11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs
Control
Autistic
Age-dependent changes in Cap and Cobalamin mRNA in Control vs. Autism
80 year old subject
HCY
5/4 FOL
CAP
11/9COB A
30/30 SAM
C
{ { {
A
C
{ { {
C
{ { {
A
Selenoproteins, mercury and redox status
Glucose is the major source of reducing power for maintaining reduced glutathione
NADP+
Glucose
NADPH Thioredoxin Reductase 6-P-gluconolactone Thioredoxin
Glucose-6-P
G6PD
GSH status
Thioredoxin reductase is a selenoprotein
NADP+
Glucose
NADPH Thioredoxin Reductase 6-P-gluconolactone Thioredoxin
Glucose-6-P
G6PD
CpG CpG CpG
G6PD gene (on)
GSH status Methionine Synthase Activity SAM SAH
DNA Demethylase
G6PD gene (off)
DNA Methyltransferase
CpG CpG CpG CH3 CH3 CH3
Hg
SULFUR AND SELENIUM AMINO ACIDS
H H3 N C CH 2 SH CYSTEINE Binding Constant = 1039 COO H3 N
H C CH 2 Se SELENOCYSTEINE Binding Constant = 1045 COO
Hg2+
(million-fold higher affinity)
From Dr. Nicholas Ralston Univ. of North Dakota
Mercury gradually migrates to highest affinity targets (i.e. selenoproteins)
Selenoproteins Thioredoxin fold proteins (dual stable thiols) Protein thiols (mono thiol sites) Thiol metabolites (GSH, cysteine)
Hg2+
Highest levels of GSH are in selenium-rich ependymal cells which are stem cells for astrocytes and neurons
Selenoprotein P Is high in Ependymal cells
Astrocyte [GSH] = 0.91 mM Neuron [GSH] = 0.21 mM
Ependymal [GSH] = 2.73 mM
Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006 Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007
Prevailing redox conditions determine the proportion of neurons vs. astrocytes which develop from neuronal stem cells
Pluripotent Stem Cells (Ependymal Cells) Oxidized State Pluripotent Stem Cells (Ependymal Cells) Normal State Pluripotent Stem Cells (Ependymal Cells) Reduced State
Less Neurons
More Astrocytes
Neurons
Astrocytes
More Neurons
Less Astrocytes
Neuronal cells
In human neuronal cells thimerosal partially inhibits the selenoprotein thioredoxin reductase with high potency, but inhibits thioredoxin with only low potency
Thioredoxin reductase
0.03
0.010
Thioredoxin
Enzyme Activity
Enzyme Activity
0.02
0.005
0.01
0.000 0
0.00
-12 -11 -10 -9 -8 -7 -6 -5 -4
0
-12 -11 -10
-9
-8
-7
-6
-5
-4
[Thimerosal] M
[Thimerosal] M
55
Thimerosal-induced reduction of GSH levels in SH-SY5Y human neuroblastoma cells
900 800 700 600 500 400 300 200 100 0
[GSH] (nmol / mg protein)
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Thimerosal] M
56
a
120
b
120 100 80 60 40 20 0 0 -11 -10 -9 -8 -7 -6 -5
MS activity pmol/min/mg protein
MS activity pmol/min/mg protein
Hydroxo-B12 Methyl-B12
100 80 60 40 20 0 0 -11 -10 -9 -8
Hydroxo-B12 Methyl-B12
-7
-6
-5
c
140
Log [Lead ] M
Hydroxo-B12 Methyl-B12
d
120
Log [Arsenic] M
Hydroxo-B12 Methyl-B12
MS activity pmol/min/mg protein
120 100 80 60 40 20 0 0 -12 -11 -10 -9
MS activity pmol/min/mg protein
100 80 60 40 20 0 0 -12 -11 -10 -9
-8
-7
-6
-5
-8
-7
-6
-5
e
100
Log [Aluminum] M
Hydroxo-B12 Methyl-B12
f
1750
Log [Mercury] M
Control Le ad Arse nic Aluminum M ercury Thime rosal
MS activity pmol/min/mg protein
[GSH] nmole/mg protein
80 60 40 20 0
1500 1250 1000 750 500 250 0
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Thimerosal] M
Genetic and Environmental Factors Can Combine to Cause Autism
Genetic Risk Factors Environmental Exposures
PON1, GSTM1
Impaired Sulfur Metabolism
Neuroinflammation
Oxidative Stress
MTRR, MTHFR, ADSL RFC, TCN2
Methionine Synthase Activity ↓
COMT, ATP10C, ADA MeCP2, ADA
↓ Receptor Phospholipid Methylation D4
MET, NLGN3/4 FMR-1, RELN
↓DNA Methylation ∆ Gene Expression Developmental Delay
↓ Neuronal Synchronization ↓ Attention and cognition
AUTISM
58
“… and a child shall lead them.”
Disorders sharing metabolic features with autism:
Attention-deficit hyperactivity disorder Alzheimer’s disease Schizophrenia Parkinson’s disease Chronic fatigue syndrome Amyotrophic lateral sclerosis Multiple sclerosis Type 2 diabetes Obesity
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