1. Redox and Methylation in the Gut, Brain and Immune System
Part I: General Principles
Richard Deth, PhD
Northeastern University
Boston, MA

2. Overview Oxidation and antioxidant metabolism
Epigenetic regulation of gene expression
Prenatal epigenetic programming (PREP)
Postnatal epigenetic programming (PEP)
Brain-specific redox features
Role of selenium and selenoproteins
Effects of heavy metals on redox and methylation
Redox signaling in the immune response 2

3. Viewing Life Through Redox Glasses
Oxidation: Loss of an electron
Reduction: Gain of an electron

4. H2S As oxygen became more plentiful,
evolution was driven by the ability
to adapt and to resist oxidation.
Earliest life appears arose at
hydrothermal vents emitting
hydrogen sulfide.
H2S
From Paul G. Falkowski; Science (2006)

5. Evolution: Development: Life: The evolved ability to resist oxidation
Death:
The inevitable outcome of oxidation
Evolution:
Gradual acquisition and manifestation of novel adaptive strategies to survive oxidation
Development:
Programmed and progressive changes in gene expression,
driven by changes in oxidation status, via epigenetic mechanisms

6. Primordial Synthesis of Cysteine
From Volcanic Gases
Methane CH3
Hydrogen sulfide H2S
Ammonia NH3
Carbon dioxide CO2
NH2CHCOOH
CH2 SH
Cysteine

7. The Glutathione Redox Equilibrium2
REDUCED GSH
OXIDIZED GSSG

8. EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO OFFSET
THE THREAT OF OXIDATION
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.

9. LANGUAGE SOCIAL SKILLS
- 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.
- Cognitive abilities of the human brain are particularly vulnerable.
LANGUAGE
SOCIAL SKILLS

10. O2 + 4H+ 2H2O + ATP formation The available level of antioxidant (GSH)
controls the level of aerobic metabolism
and ATP formation = Redox Signaling
Selenoproteins
Antioxidant
Supply
Glucose
NADPH
Glutathione
NADH+
O2 + 4H+
2H2O + ATP
formation
Reactive
Oxygen
Species
ROS
[Antioxidant]
Oxidative
Cellular
Damage
No Damage
Glutathionylation
of Complex I

11. Mitochondrial respiration is a primary
source of reactive oxygen species
Oxygen
ATP +
H2O
Reactive
Oxygen
Species
(ROS)
From: Kiley P J & Storz G; 2004 11

12. Cysteine is taken up from the GI tract and is
distributed to the body and the brain
GSH

13. The brain extracellular environment (CSF) has
more than 100-fold less cysteine than plasma!!
Blood-Brain
Barrier
BRAIN
BLOOD
Neurons
Astrocytes
[GSH] = 0.21mM
[GSH] = 0.91mM
[GSH] = 3.5 μM
[CYS]
[CYS] =282 μM
[CYS]
[CysGly]
[GSH]
CSF
[GSH] = 0.3 μM
[CYS] = 2.2 μM
12-fold lower
125-fold lower
Data from: Castagna et al. Neurology, 45: (1995) and Sun et al. J Biol Chem, 281: (2006). 13

14. Cysteine for glutathione synthesis can be provided by either
transsulfuration of homocysteine or by uptake from outside the cell
NEURONAL
CELL 14 14

15. REDOX STATUS: GSH GSSH Methylation Status: SAM SAH ~ 200 Reactions
Nitric Oxide
Synthesis
Phospholipid
DNA/Histone
Gene
Expression
Arginine
Membrane
Properties
Creatine
Cognitive
Status
Energy
Catecholamine
Serotonin
Melatonin
Sleep

16. Methionine synthase provides redox-sensitive
global coordination of metabolism: HOMEOSTASIS

17. Methylation of DNA and histones is fundamental to
epigenetic regulation of gene expression during development

18. Regulation of gene expression during development
Transcription Factor Regulation:
Growth Factors
Start site for
mRNA synthesis
Neurotransmitters
Hormones TF
CpG
CpG
TF binding region
Gene sequence
DNA
RNA
polymerase
mRNA
Transcription
Translation
Protein
(e.g. enzyme)
Epigenetic Regulation:
SAM Me Me
MBDP
(e.g. MeCP2)
HMT
Histone
proteins Me Me
SAM
DNMT
CpG
CpG Me Me X
No mRNA
TF binding region
DNA
DNA + Histone = Heterochromatin
Genes are silenced and transcription is blocked

19. Epigenetic changes in gene expression are the
Primary mechanism underlying development

20. Epigenetic marks change in response to the environment and
contribute to adaptive capabilities gained through evolution

21. Essentially:
Epigenetics = A memory system
linked to gene expression
responsive to redox status
driver of development
active in all cell types
active at all ages
capable of transgenerational effects
Agents which interrupt antioxidant
and/or methylation pathways will
interfere with the many roles of
epigenetic regulation.

22. Emotional Experiences
PrEP
PEP
Postnatal
Epigenetic
Programming
Prenatal
Epigenetic
Programming
Maternal Metabolism
Maternal Nutrition
Maternal
Toxic Exposures
Placental Function
Genetic Factors
Breast Milk (Formula)
Toxic Exposures
Physical Experiences
Emotional Experiences
BIRTH

23. ↑ Metabolic Activity ↓Antioxidant Availability
(i.e. cysteine and GSH)
Metabolic Activity
(Antioxidant demand)
Homeostatic Equilibrium
↑ Metabolic Activity
(Higher antioxidant demand)
↓Antioxidant Availability
(Low cysteine and GSH)
Adaptive
Epigenetic
Changes
Oxidative Stress →

24. Odds of autism decrease with increasing duration of breastfeeding

25. GI tract absorption of cysteine is critical for
postnatal epigenetic programming (PEP)

26. Transsulfuration Pathway
REDOX & METHYLATION PATHWAYS
Transsulfuration Pathway
Redox
Buffering
Glutathione (GSH)
γ-Glutamylcysteine
Cysteine
Methionine
Cycle
D4-Receptor
PLM Cycle
Cystathionine
Adenosine
Adenosine
D4-SAH
D4-HCY
HCY
SAH
( - )
Methyl-THF
Methyl-THF
Phospholipid
Methylation
Methionine
Synthase
> 150
Methylation
Reactions
THF
THF
THF
D4-SAM
D4-MET
MET
SAM
PP + Pi
ATP
PP + Pi
ATP
Dopamine (Attention)

27. D4HCY D4SAM D4SAH D4MET NEURON
Neurons have impaired transsulfuration and low GSH levels
that depend upon growth factor-stimulated cysteine uptake
Methionine
Synthase
HCY
MET
SAH
SAM
>1,000
Methylation
Reactions
ATP
PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
D4HCY
D4SAM
D4SAH
D4MET
Phospholipid
Dopamine
( - )
PI3-kinase
( + )
PARTIALLY BLOCKED IN NEURONAL CELLS
EAAT3
Astrocytes
Cysteinylglycine
GSSG
Neurotrophic
Growth
Factors
NEURON
Cystine 27 27 27

28. 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 Feb;230(2):

29. Neurotrophic growth factors increase
REDOX SIGNALING:
Neurotrophic growth factors increase
EAAT3-mediated uptake of cysteine.
Leading to:
-Increased GSH/GSSG
-Increased methionine synthase activity
-Increased DNA methylation and
epigenetic consequences 29

30. Growth factor regulation of cysteine uptake is a powerful
mechanism to regulate redox and methylation in the brain
Glial Cells
(Astrocytes)
Neurotrophic
Growth Factors
Cysteine
Cysteinylglycine
GSH
EAAT3
( + )
GSSG
GSH
PI3-kinase
γ-Glutamylcysteine
Cysteine
EAAT3
Cystathionine
Adenosine
HCY
SAH
( - )
MethylTHF
Methionine
Synthase
>150
Methylation
Reactons
THF
MET
SAM
ATP
PP+Pi 30 30 30

31. IGF-1 stimulates methionine synthase activity and
increases DNA methylation in human neuronal cells

32. By increasing cysteine uptake, neurotrophic growth factors can
increase methionine synthase activity and regulate gene expression
Glial Cells
(Astrocytes)
Neurotrophic
Growth Factors
Cysteine
Cysteinylglycine
GSH
EAAT3
GSSG
GSH
( + )
PI3-kinase
γ-Glutamylcysteine
( + )
Epigenetic regulation
of gene expression
EAAT3
Cysteine
Cystathionine
Adenosine
( + )
HCY
SAH
( - )
MethylTHF
DNA
Methylation
Methionine
Synthase
THF
MET
SAM
ATP
PP+Pi 32 32 32

33. REDOX SIGNALING: Tumor necrosis factor–alpha (TNF-alpha),
a pro-inflammatory cytokine, decreases
EAAT3-mediated uptake of cysteine.
Leading to:
- Decreased methionine synthase mRNA
Increased transsulfuration
TNF-alpha levels are elevated in
brain and CSF of autistic subjects 33

34. TNF-alpha decreases cysteine uptake

35. TNF-alpha causes a large and prompt
decrease in methionine synthase mRNA

36. Despite inhibition of cysteine uptake, cysteine and
GSH levels do not decrease after TNF-alpha,
indicating that it increases transsulfuration

37. TNF-alpha decreases cysteine uptake and decreases
methionine synthase activity, but increases transsulfuration
Glial Cells
(Astrocytes)
TNF- alpha
Cysteine
Cysteinylglycine
GSH
EAAT3 ( )
GSSG
GSH
γ-Glutamylcysteine ( )
Epigenetic regulation
of gene expression
Cysteine
( + )
Cystathionine
Adenosine
HCY
SAH ( )
MethylTHF
↓ DNA
Methylation
Methionine
Synthase
THF
MET
SAM
ATP
PP+Pi 37 37 37

38. NEUROTROPHIC GROWTH FACTORS
EAAT3-Mediated Influx
GROWTH FACTORS
Redox Status
Epigenetic
Regulation
( > 150 Reactions )
Transsulfuration
TNF-alpha (Inflammatory Cytokine)
GSH
GSSG
METHYLATION
Neural Network
Synchronization
Myelination
CYSTEINE
( + )
( - )
Methionine
Synthase
HCY
MET
SAH
SAM

39. Selenium and selenoproteins in redox regulation39

40. Oxygen, sulfur and selenium are chemically similar in the periodic table.

41. (Reducing Equivalent)
Water
Oxygen
e.g ROS
Reducing equivalents
are passed from
selenium to sulfur and
from sulfur to oxygen e-
Sulfur
e.g. GSH e-
NADPH
Selenium
e.g. Thioredoxin
Reductase
Hydrogen
(Reducing Equivalent)
Illustrations from: Bentor, Yinon. Chemical Element.com

42. Reducing electrons move from NADPH through the selenoprotein
thioredoxin reductase to thioredoxin and glutathione
Thioredoxin Reductase
Electron flow

43. Selenocysteine-based
Selenoproteins provide antioxidant electrons e- e-
Selenocysteine-based
Redox Proteins
(TrxR ,TGR, GPx) e-
Cysteine-based
Redox Proteins
(Trx , Prx)
Glucose
NADPH e- e- e- GR e-
-Reduction of oxidized proteins
and phospholipids.
-Reduction of glutathionylated proteins e- e-
GSSG
GSH
Grx
GR = glutathione reductase Trx = thioredoxin
GSH = reduced glutathione TrxR = thioredoxin reductase
GSSG = oxidized glutathione TGR = thioredoxin-glutathione reductase
GPx = glutathione peroxidase Prx = peroxiredoxin
Grx = glutaredoxin e- = electron

44. The selenoprotein Thioredoxin reductase (TrxR) is directly
or indirectly responsible for keeping vitamin E, ascorbic acid,
glutathione, thioredoxin and ubiquinone reduced.

45. Glucose is the major source of reducing power
for maintaining reduced glutathione
Selenoprotein
NADP+
NADPH
Thioredoxin Reductase
Glucose
Glucose-6-P
6-P-gluconolactone
G6PD
Thioredoxin
CpG CpG CpG
G6PD gene (on)
GSH status
DNA
Demethylase
Methionine
Synthase
Activity
G6PD gene (off)
CpG CpG CpG
DNA Methyltransferase
SAM
SAH
CH3
CH3
CH3 45

46. 46

47. 47

48. From Dr. Nicholas Ralston Univ. of North Dakota
SULFUR AND SELENIUM AMINO ACIDS H H H N C
COO H N C
COO 3 3 CH CH 2 2 Se SH
CYSTEINE
SELENOCYSTEINE
Although structurally analogous to cysteine, selenocysteine is genomically and biochemically unique. Selenocysteine is encoded by UGA, which is normally a stop codon, but in selenoproteins a specific structural element changes the code to a selenocysteine insertion element. Selenocysteine is synthesized de novo during each cycle of protein synthesis and cannot be reutilized in subsequent cycles of protein synthesis. Selenocysteine’s selenium is ionized at physiological pH while the sulfur of cysteine is protonated. Selenocysteine also has a far broader redox potential than cysteine, making it capable of catalyzing biochemical reactions cysteine could never accomplish.
Binding Constant = 1039
Binding Constant = 1045
Hg2+
(million-fold higher affinity)
From Dr. Nicholas Ralston Univ. of North Dakota

49. 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+ 49

50. 50

51. Selenoprotein functions relevant to autism
( - )
Selenocysteine
Hg2+
Selenoprotein Activities
Redox Regulation and Antioxidant Activity
(e.g. TrxR)
Thyroid Hormone
Synthesis
(e.g. DIO)
ROS Inactivation and Oxidation Repair
(e.g. GPx)
Intracellular Redox Status
Methionine Synthase
Activity
Epigenetic
Regulation of
Gene Expression
DNA and Histone
Methylation
Brain
Development
*Mercury inhibition of selenoproteins could disrupt redox regulation and
epigenetic regulation during brain development

52. Highest levels of GSH are in selenium-rich ependymal cells
Astrocyte [GSH] = 0.91 mM
Neuron [GSH] = 0.21 mM
Ependymal [GSH] = 2.73 mM
Selenoprotein P
Is high in
Ependymal cells
Highest levels of GSH are in selenium-rich ependymal cells
which are stem cells for astrocytes and neurons
Sun et al. J BIOL CHEM. VOL. 281, pp –17431, 2006
Scharpf et al. J. NEURAL TRANS.VOL 114, , 2007 J

53. Adult neurogenesis occurs in the subventricular zone (SVZ)
of the cortex and the subgranular zone (SGZ) of the hippocampus
Vescovi et al. Nature Reviews Cancer 6, 425–436 (June 2006)

54. 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

55. GSH Does Redox Control Development Via Epigenetic Effects? ∆ Gene
Eggs are rich
In cysteine
Sperm are rich
in selenium
GSH
∆ DNA and Histone
Methylation
∆ Gene
Expression
Does Redox Control
Development Via
Epigenetic Effects?
juanv.wordpress.com/ 55

56. Neuronal cells 56

57. REDOX SIGNALING IN THE
IMMUNE SYSTEM: 57

58. Redox signaling plays a central role in the immune response
Dendritic cells release GSH,
similar to astocytes in brain
Effector T-cells take up cysteine,
similar to neurons in brain
Antibody
production
Antigen
EAAT3 provides
Cysteine uptake
Regulatory T-cells compete
with Teff for cysteine
and restrict the immune response

59. EAAT3 is expressed in T cell lymphocytes
and highest expression is in Treg cells

60. Autoimmune-prone SJL/J mice have lower EAAT3
expression in mixed T cell lymphocytes

61. Autoimmune-prone SJL/J mice have lower levels of GSH
in brain, unaffected by postnatal thimerosal treatment

62. Autoimmune-prone SJL/J mice have lower methionine synthase
activity in brain, unaffected by postnatal thimerosal treatment

63. ACKNOWLEDGEMENTS Brain Samples: Autism Tissue Program
Harvard Brain Tissue Resource Center
Tissue Resource Center (Australia)
Stanley Medical Research Foundation
and donor families.
Collaborators:
Antonio Persico
Suzanne De la Monte
Hamid Abdolmaleky
Sultan Qaboos University
Mostafa Waly and Yahya Al-Farsi
Grant Support:
Autism Research Institute
SafeMinds
National Autism Association
Autism Speaks