Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism
Richard Deth, PhDNortheastern 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
Earliest life appears to have arisen at hydrothermal vents emitting hydrogen sulfide and other gases at high temperature and pressure
H2O
H2S
Methane CH3
Hydrogen sulfide H2SAmmonia NH3
Carbon dioxide CO2
NH2CHCOOHCH2
SH
Cysteine
Primordial Synthesis of CysteineFrom Volcanic Gases
NH2CHCOOH
CH2
SH
NH2CHCOOH
CH2
SH
+
NH2CHCOOHCH2
S
NH2CHCOOHCH2
S+ 2 H+
Cysteine Disulfide
Two AntioxidantReducing Equivalents
Cysteine can function as an antioxidant
Two Cysteines
O2
O2
O2
O2
GeneticMutation
NovelAntioxidantAdaptation
Evolution = Adaptation to threat of oxidation
Adaptive features of sulfur metabolism=
Evolution = Metabolic Adaptations
to an Oxygen Environment
Figure from Paul G. FalkowskiScience 311 1724 (2006)
EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO ENVIRONMENTAL THREATS
The ability to controloxidation is at thecore of evolution
Each addition isstrengthened because
it builds on thesolid 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 andif there is a significant changes in the environment.
Humans cognitive abilities are particularly vulnerable.
LANGUAG
E
SOCIAL SKILLS
NORMALREDOX
BALANCE
Redox Buffer Capacity
[Glutathione]
Oxygen Radicals
OxygenRadicals
Redox Buffer Capacity
OXIDATIVE STRESS
Methylation
GeneticRisk Factors
Heavy Metals+
Xenobiotics
OxidativeMetabolism
Neuronal Synchronization
Neuronal Degeneration
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
( - )
GSSG
Dietary protein
Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell
GlutathioneSynthesis
REDOXSTATUS:
GSHGSSH
MethylationStatus:SAMSAH
~ 200 Methylation
Reactions
Nitric OxideSynthesis
PhospholipidMethylation
DNA/HistoneMethylation
GeneExpression
ArginineMethylation
MembraneProperties
CreatineSynthesis
CognitiveStatus
EnergyStatus
CatecholamineMethylation
SerotoninMethylation
Melatonin
Sleep
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
( - )
GSSG
Dietary protein
During oxidative stress methionine synthase is turned off,allowing more homocysteine to flow toward GSH synthesis,
while methylation activity is decreased
GlutathioneSynthesis
OXIDATIVESTRESS
Oxidative Stress
GSHGSSG = 30 GSH
GSSG = 10Normal
Redox Setpoint
- Survival mode
- Loss of normal function
- Impaired methylation
Infection, allergy,trauma, chronic illness
Recovery:Adaptive responsesto oxidative stress
InflammatoryState
Inflammation is a metabolic state of oxidative stress, normally occurring in response to environmental challenges
Oxidative Stress
GSHGSSG = 30 GSH
GSSG = 10Normal
Redox Setpoint
↑ Inflammatory Diseases:
- Alzheimer’s disease- Parkinson’s disease- Diabetes- Heart Failure
Aging
Aging is associated with increased oxidative stress, as adaptive responses fail to restore normal redox status
Oxidative Stress
GSHGSSG = 30 GSH
GSSG = 10Normal
Redox Setpoint
- Survival mode
- Loss of normal function
- Impaired methylation
- Autism??
Heavy Metal and Xenobiotic Exposure Inflammatory
State
Exposure to persistent environmental toxins promotes oxidative stress and impairs the ability to recover
38%↓
28%↓
36%↓
Autism is associated with oxidative stress and impaired methylation
The Brain Compartment (CSF) has low Thiol levels and maintains an Oxidative Stress environment;
Astrocytes provide Cysteine to Neurons for survival
Blood-BrainBarrierBRAIN BLOOD
[GSH] = 8μM
[CYS] =200μM
[GSH] = 0.91mM[GSH] = 0.21mM
[CYS] = 2 μM
[CYS]
[CYS]
[GSH] = 1 μMCSF
Neurons Astrocytes
[GSH][CysGly]
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
( - )
PI3-kinase
( + )EAAT3
HealthyGlial Cells
(Astrocytes)Cysteinylglycine GSH
GSSG
Growth Factors
Neurons obtain cysteine from GSH released by Glial cells,via a growth factor-controlled transporter (EAAT3)
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
( - )
PI3-kinase
( + )
PARTIALLY BLOCKED IN NEURONAL CELLS
EAAT3
HealthyGlial Cells
(Astrocytes)Cysteinylglycine GSH
GSSG
Growth Factors
Transsulfuration of homocysteine (HCY) to cysteine is restricted inhuman neuronal cells, increasing importance of cysteine uptake
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
GSCbl
Cysteine
( - )
PI3-kinase
( + )
PARTIALLY BLOCKED IN NEURONAL CELLS
MeCbl
EAAT3
HealthyGlial Cells
(Astrocytes)Cysteinylglycine GSH
SAMGSSG
H2S
OHCbl
Growth Factors
Methionine synthase in human neuronal cells requires methylB12 (MeCbl),whose synthesis is glutathione-dependent
Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.
Levels of cystathionine are markedly higher inhuman cortex than in other species
In neurons, D4 dopamine receptors carry out phospholipid methylation,which requires methionine synthase to supply methyl groups
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
D4HCY
D4SAM
D4SAH
D4METATPPP+Pi
MethylTHF
THF
PhospholipidMethylation
Adenosine
Dopamine
Cysteine
( - )
PI3-kinase
( + )
PARTIALLY BLOCKED IN NEURONAL CELLS
EAAT3
HealthyGlial Cells
(Astrocytes)Cysteinylglycine GSH
GSSG
Growth Factors
GSCbl
MeCbl
SAMOHCbl
25
CH3
DOPAMINE
DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION
MethylfolateMethionineSynthase
7-repeats
2 or 4-repeats
Methionine SynthaseStructure and function
Brain levelsAcross the lifespanIn autism
Methionine synthase has five domains + cobalamin (Vitamin B12)Domains alternate interacting with cobalamin during turnover
SAM Domain
CobalaminDomain Cap
Domain
Cobalamin(vitamin B12)
SAM Domain
CobalaminDomain Cap
Domain5-Methyl THF Domain
HCY Domain
Cobalamin(vitamin B12)
1
23
HCY FOL CAP COB SAM
187 bp 197 bp 419 bp
3' 5'
Exon 19 252420
188 bp 122 bp
21 22 23
HCY FOL CAP COB SAM
187 bp 197 bp 419 bp
3' 5'
Exon 19 252420
188 bp 122 bp
21 22 23
Decrease of Cob domain mRNA with increasing age, 40 subjects
0 10 20 30 40 50 60 70 80 90 1000
100
200
300
400
500
600T1/2fast = 3.4 years
T1/2slow = 29.4 yearsR2 = .91
Age (years)
MS
Cob
mR
NA
(arb
itrar
y un
its)
Decrease of Cap domain with increasing age, 40 subjects
0 10 20 30 40 50 60 70 80 900
100
200
300
400
500
600
700T1/2fast = 2.2 yearsT1/2slow = 20 years
R2 = .94
Age (years)
MS
Cap
mR
NA
(arb
itrar
y un
its)
CAP Domain is present in MS mRNA from 24 y.o. subject
HCY FOL CAP COB SAM
CAP Domain is absent from methioninesynthase mRNA in elderly human cortex
HCY FOL CAP COB SAM
80 year old subject
Age-dependent decrease in the ratio of Cap to Cobalamin mRNA
HCY FOL CAP COB SAM
80 year old subject
Under 20
Over 6
00.0
0.5
1.0
1.5M
S m
RN
A C
ap/C
ob R
atio
Cap Domain Exons 19-21
Site of alternative splicing by mRNA-specific adenosine deaminase
Cap Domain Absent
Cap Domain Present
HCY FOL COB SAM
Pre-mRNA mRNA
Alternative Splicing of MS Pre-mRNAleads to age-dependent exon skipping
Exons 16-18 aredeleted in
fetal human brain
115
180
84
115
180
84
MS exists as two lower MW bands in SH-SY5Y cells
Normal full size MW = 140 kDa
125 kDaExons 16-18are absent
110 kDaExons 16-20are absent
DNA
RNA(exons only)
Pre-mRNA(introns + exons)
Protein
Transcription
Splicing
Translation
B12 Cofactor
DNA
RNA(exons only)
Pre-mRNA(introns + exons)
Protein
Transcription
Splicing
Translation
DNA
RNA(exons only)
Pre-mRNA(introns + exons)
Protein
Transcription
Splicing
Translation
B12 Cofactor
Methionine synthase activity can be regulated via multiple levels of control in response to oxidative stress
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
0 10 20 300
100
200
300
400
Autistic
ControlsT1/2 = 2.7 yrs r2 = 0.94
Age (years)
MS
Cap
leve
ls (a
rbitr
ary
units
)
Paired comparisons of CAP domain mRNA to age-matched controls
(Same samples as Vargas et al. 2005)
0
100
200
300
400
11-15 yrs
26-30 yrs21-25 yrs
1-5 yrs6-10 yrs
16-20 yrs
Control Autistic
Met
hion
ine
Synt
hase
CAP
dom
ain
mR
NA
(arb
itrar
y un
its)
Paired comparisons of Cob domain mRNA to age-matched controls
(Same samples as Vargas et al. 2005)
0
100
200
300
400
11-15 yrs
26-30 yrs21-25 yrs
1-5 yrs6-10 yrs
16-20 yrs
Control Autistic
Met
hion
ine
Synt
hase
CO
B d
omai
n m
RN
A(a
rbitr
ary
units
)
Age-dependent changes in Cap and Cobalamin mRNA in Control vs. Autism
HCY FOL CAP COB SAM
80 year old subject{ { {C A C A C A
5/4 11/9 30/30{ { {C A C A C A
5/4 11/9 30/30{ { {C A C A C A
5/4 11/9 30/30
C A C A C A
5/4 11/9 30/30
Selenoproteins, mercury and redox status
G6PD
NADP+
6-P-gluconolactone Glucose-6-P
NADPH Thioredoxin Reductase
Thioredoxin
GSH status
Glucose is the major source of reducing powerfor maintaining reduced glutathione
Glucose
CpG CpG CpG G6PD gene (on)
G6PD
NADP+
6-P-gluconolactone Glucose-6-P
NADPH Thioredoxin Reductase
Thioredoxin
DNA MethyltransferaseCpG CpG CpG
G6PD gene (off)
CH3 CH3 CH3
MethionineSynthaseActivity
SAMSAH
GSH status
DNADemethylase
Thioredoxin reductase is a selenoprotein
Glucose
Hg
CH
SH
N
H
H COOC3
2CH
Se
N
H
H COOC3
SELENOCYSTEINE
2
CYSTEINE
SULFUR AND SELENIUM AMINO ACIDS
From Dr. Nicholas Ralston Univ. of North Dakota
Hg2+Binding Constant = 1045Binding Constant = 1039
(million-fold higher affinity)
Selenoproteins
Thioredoxin fold proteins(dual stable thiols)
Protein thiols (mono thiol sites)
Thiol metabolites (GSH, cysteine)
Hg2+
Mercury gradually migrates to highestaffinity targets (i.e. selenoproteins)
Astrocyte [GSH] = 0.91 mM
Neuron [GSH] = 0.21 mM
Ependymal [GSH] = 2.73 mM
Selenoprotein P Is high inEpendymal cells
Highest levels of GSH are in selenium-rich ependymal cellswhich are stem cells for astrocytes and neurons
Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007
Pluripotent Stem Cells(Ependymal Cells)
Oxidized State
MoreAstrocytes
LessNeurons
Pluripotent Stem Cells(Ependymal Cells)
Normal State
AstrocytesNeurons
Pluripotent Stem Cells(Ependymal Cells)
Reduced State
LessAstrocytes
MoreNeurons
Prevailing redox conditions determinethe proportion of neurons vs. astrocyteswhich develop from neuronal stem cells
Neu
rona
l cel
ls
55
In human neuronal cells thimerosal partially inhibits the selenoprotein thioredoxin reductase with high potency,
but inhibits thioredoxin with only low potency
Thioredoxin
-12 -11 -10 -9 -8 -7 -6 -5 -40.00
0.01
0.02
0.03
0[Thimerosal] M
Enzy
me
Activ
ity
Thioredoxin reductase
-12 -11 -10 -9 -8 -7 -6 -5 -40.000
0.005
0.010
0[Thimerosal] M
Enzy
me
Activ
ity
56
Thimerosal-induced reduction of GSH levelsin SH-SY5Y human neuroblastoma cells
-12 -11 -10 -9 -8 -7 -6 -500
100200300400500600700800900
Log [Thimerosal] M
[GSH
](n
mol
/ m
g pr
otei
n)
0
20
40
60
80
100
120
Methyl-B12
0 -11 -10 -9 -8 -7 -6 -5
Log [Lead ] M
Hydroxo-B12
MS
activ
itypm
ol/m
in/m
g pr
otei
n0
20
40
60
80
100
120
Methyl-B12
0 -11 -10 -9 -8 -7 -6 -5
Hydroxo-B12
Log [Arsenic] M
MS
activ
itypm
ol/m
in/m
g pr
otei
n
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100
120
140Hydroxo-B12Methyl-B12
Log [Aluminum] M
MS
activ
itypm
ol/m
in/m
g pr
otei
n
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100
120Hydroxo-B12Methyl-B12
Log [Mercury] M
MS
activ
itypm
ol/m
in/m
g pr
otei
n
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100Hydroxo-B12Methyl-B12
Log [Thimerosal] M
MS
activ
itypm
ol/m
in/m
g pr
otei
n
0
250
500
750
1000
1250
1500
1750ControlLeadArsenicAluminumMercuryThimerosal
[GSH
]nm
ole/
mg
prot
ein
a b
c d
e f
58
Genetic and Environmental Factors Can Combine to Cause Autism
FMR-1, RELN
MeCP2, ADA
RFC, TCN2
COMT, ATP10C, ADA
PON1, GSTM1
MET, NLGN3/4
Genetic Risk Factors Environmental Exposures
Impaired Sulfur Metabolism
Oxidative Stress
D4 Receptor Phospholipid Methylation
Neuronal Synchronization
↓Attention and cognition
Methionine Synthase Activity
DNA Methylation
Gene Expression
Developmental Delay
AUTISM
↓
AUTISM
FMR-1, RELN
MeCP2, ADA
RFC, TCN2
COMT, ATP10C, ADA
PON1, GSTM1
MET, NLGN3/4
MTRR, MTHFR, ADSL
↓
↓ ↓
Neuroinflammation
“… and a child shall lead them.”Disorders sharing metabolic features with autism:
Attention-deficit hyperactivity disorderAlzheimer’s diseaseSchizophreniaParkinson’s diseaseChronic fatigue syndromeAmyotrophic lateral sclerosis Multiple sclerosisType 2 diabetesObesity
Thanks for your Research Support!!
Autism Research Institute
SafeMinds
Cure Autism Now
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