Methionine Synthase in Neuronal Cells:

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Methionine Synthase in Neuronal Cells A
MethylB12-dependent Redox Sensor Richard C.
Deth Northeastern University
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• Overview
• Sulfur metabolism and oxidative stress
• Methionine synthase and methylcobalamin
• D4 dopamine receptor-mediated PLM
• Autism, ADHD, Alzheimers and Parkinsons

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Metabolic Adaptations to an Oxygen
Environment Are Closely linked to Evolution (and
survival)
From Dr. Paul Falkowski, Science 311 1724 (2006)
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Mitochondria -Use oxygen (O2) to make
ATP -Efficiency is only 95 99 -Some
oxygen is released as reactive oxygen species
or ROS hydrogen peroxide (H2O2) superoxide
anion (O2-.) - ROS must be inactivated by
glutathione
ATP H20
ADP O2 H
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Two Primary Roles of Sulfur Metabolism
SULFUR METABOLISM
Cellular Oxidative Status
Cellular Methylation Status
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Oxygen Radicals
Oxidative Metabolism
Genetic Risk Factors
Oxygen Radicals
Redox Buffer Capacity
Mercury Xenobiotics
Redox Buffer Capacity
OXIDATIVE STRESS
Methylation
NORMAL REDOX BALANCE
Neuronal Degeneration
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Transsulfuration
Oxidative Stress Response
Insulin/IGF-1
PI3-kinase
Glutathione
EAAT3
?-Glutamylcysteine
Glutamate
Glutamate
EAAT3
Cysteine sulfinate
Cysteine
Cysteine
Cysteine
Neurons
Cystathionine
AMP
Cysteine
methylfolate
HCY
Adenosine
methylB12
Inosine
Cysteinylglycine
Diet
MET
SAH
ATP
SAM
GSH
Methylation
Astrocytes
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Cobalamin oxidation and enzyme cleavage increase
diversion of HCY to glutathione during oxidative
stress
Redox
Methylation
Glutathione
S-Adenosylmethionine
Oxidative Stress Response
Cysteine sulfinate
Cysteine
Cysteine Dioxygenase
Methionine
Cystathionine
Ubiquitin-dependent Proteolysis
Cystathionine ?-Synthase
Methionine Synthase
HCY
CBS
Adenosine
Ubiquitin-dependent Proteolysis
? GSH ?
S-Adenosyl- homocysteine
CBS
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Cobalamin oxidation and enzyme cleavage increase
diversion of HCY to glutathione during oxidative
stress
Redox
Methylation
Glutathione
S-Adenosylmethionine
Oxidative Stress Response
Cysteine sulfinate
Cysteine
Cysteine Dioxygenase
Methionine
Cystathionine
Cbl(I)?Cbl(II)
Ubiquitin-dependent Proteolysis
Cystathionine ?-Synthase
Methionine Synthase
HCY
Oxidative Stress
CBS
Adenosine
Ubiquitin-dependent Proteolysis
? GSH ?
S-Adenosyl- homocysteine
CBS
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Methionine synthase has five domains cobalamin
(B12)
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Role of B12 Cobalamin in Methionine Synthase
From McCaddon et al. Neurology 58 1395-99 (2002)
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Methionine Synthase has a lower MW in SH-SY5Y
cells What is the missing part??
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Cobalamin oxidation and enzyme cleavage increase
diversion of HCY to glutathione during oxidative
stress
Redox
Methylation
Glutathione
S-Adenosylmethionine
Oxidative Stress Response
Cysteine sulfinate
Cysteine
Cysteine Dioxygenase
Methionine
Cystathionine
Cbl(I)?Cbl(II)
Ubiquitin-dependent Proteolysis
Cystathionine ?-Synthase
Methionine Synthase
HCY
CBS
MS
Adenosine
Ubiquitin-dependent Proteolysis
? GSH ?
Ubiquitin-dependent Proteolysis ???
? GSH ?
S-Adenosyl- homocysteine
CBS
MS
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Methionine Synthase requires methylB12 if the
SAM domain is missing
Oxidative Stress
HCY
5-methylTHF
Cleavage
B12
MeB12
Oxidized MeB12
Fresh MeB12
SAM-binding domain that rescues oxidized B12
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Synthesis of bioactive methylcobalamin
(methylB12) requires glutathione and SAM
Hydroxycobalamin Cyanocobalamin
GSH
GSH
Glutathionylcobalamin
SAM
5-MethylTHF
Methylcobalamin
Methionine Synthase
Homocysteine
Methionine
D4RMET
D4RHCY
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After depletion of GSH, MethylB12 is required for
Methionine Synthase activity in SH-SY5Y Neuronal
Cells
750
MeCblSAMGSH
MeCbl
MeCblSAM
500
MeCblGSH
MeCblSAMGSH
Methionine Synthase Activity
pmol/min/mg protein
250
0
BSO
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MethylB12 is Particularly Important for
Methionine Synthase Activity in the Cortex
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SAM Domain RNA is Present in SY5Y Human
Neuroblastoma Cells
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CAP Domain RNA is Almost Absent in SY5Y Human
Neuroblastoma Cells
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Methionine Synthase requires more methylB12 if
the CAP domain is missing
Oxidative Stress
HCY
5-methylTHF
RNA Editing
Oxidized MeB12
MeB12
More MeB12
Normal form of MS in brain??
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Methionine Synthase The Redox Sentinel
Three mechanisms for regulating Methionine
Synthase

• 1. Oxidation of Cobalamin Cob(I) to Cob(II)
• Can occur during each reaction cycle, but usually
  only 1 out of 100 or less
• Increased during cellular oxidative stress
• Increased by environmental toxins and their
  metabolites
• Needs either SAM-dependent repair or methylB12
  replacement
• Enzyme stays Off for a few seconds until
  cobalamin is repaired
• Allows a few seconds of increased cysteine
  and GSH synthesis
• 2. Removal of the SAM-dependent repair domain
• - Promoted by sustained oxidative stress
• Carried out by the ubiquitin/proteasome system
• Makes enzyme depend upon methylcobalamin and
  glutathione
• Enzyme stays Off until there is enough
  glutathione
• 3. Removal of Cap domain by RNA editing
• Prominent in neuronal cells
• Increased by oxidative stress?

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• Major Causes of Oxidative Stress
• Genetic vulnerability factors in conjunction with
• - aging gradual decrease in glutathione and
  IGF-1
• heavy metals food, air, water, vaccines
  (Thimerosal)
• xenobiotics industrial, agricultural, domestic
  exposures

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Hallmark Symptoms of Autism - Impaired
reciprocal social interaction - Impaired
attention - Impaired communication -
Stereotypical behaviors
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Plasma levels of methionine cycle-related
metabolites are abnormal in autism in
association with oxidative stress
?25
?45
3-fold?
Data from Dr. Jill James
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IGF-1-stimulated PLM is inhibited by Hg2 and
Pb2
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Thimerosal potently inhibits phospholipid
methylation
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() with SAM added
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Addition of GSH Reverses Thimerosal Inhibition of
Methionine Synthase in SH-SY5Y Neuronal Cells
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Thimerosal lowers GSH levels in neuronal cells
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Plasma levels of methionine cycle-related
metabolites are abnormal in autism in
association with oxidative stress
?25
?45
3-fold?
Data from Dr. Jill James
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Methylation-promoting treatments improve plasma
levels of metabolites in autism
Folinic acid Betaine
Add MethylB12
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The D4 Dopamine Receptor Requires Methionine
Synthase to carry out Phospholipid Methylation
Hydroxycobalamin Cyanocobalamin
GSH
GSH
Glutathionylcobalamin
SAM
5-MethylTHF
Methylcobalamin
Methionine Synthase
Homocysteine
Methionine
D4RMET
D4RHCY
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Molecular Model of the Dopamine D4 Receptor
Dopamine
Methionine 313
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The D4 Receptor Cycle of Phospholipid Methylation
5-methylTHF
Adenosine
D4 HCY
Methionine Synthase
Vit B12
D4 SAH
D4 MET
ATP
( - )
D4 SAM
? Membrane fluidity
? Protein function
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Structural features of the dopamine D4 receptor
Seven repeats are associated with increased risk
of ADHD
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Dopamine-stimulated phospholipid methylation is
reduced for the 7-repeat form of the D4 Receptor
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THE MOLECULAR MECHANISM OF ATTENTION A HYPOTHESIS
de novo Purine Synthesis
Single-carbon Folate Pathway
5-methylTHF
()
(-)
D4R-mediated Phospholipid Methylation
Dopamine Stimulation
Adenosine Inhibition
Increased Local Membrane Fluidity
Modulation of Receptors, Channels and Transporters
?Gamma frequency oscillations
ATTENTION
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Ion Channels and Ion Transport Proteins can
be Considered as Elements in an Electrical
Circuit
Voltage-gated Channels
Na or Ca2 Pumps
Ligand-gated Channels
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Combined theta and gamma oscillations in neuronal
firing
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Increased gamma frequency activity in response to
a visual task at eight months of age vs. six
months
Csibra et al. Science 290 1582-1585 (2000)
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Dopamine causes an increase in gamma frequency in
a patient with Parkinsonism
Blue with dopamine (l-DOPA)
Engel et al. Nature Rev. 2005
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Gamma frequency oscillations promote
effective interaction between brain regions
with dopamine
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Brocas area
Wernickes area
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Science, Vol 308, Issue 5730, 1856-1858 , 24 June
2005
News Focus NEUROLOGYAutistic Brains Out of
Synch? Ingrid Wickelgren Autism researchers are
hot on the idea that autism results from abnormal
communications between brain regions rather than
a broken part of the brain
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AUTISM
Impaired Synchronization