Transcript DETH DS Lecture

Down Syndrome Trisomy 21:
Understanding the Problem
Richard C. Deth, PhD
Department of Pharmaceutical Sciences
Northeastern University
Key Points:
• DS (trisomy of Chr21) is a result of Chr21
nondisjunction during meiosis (cell division of egg/sperm)
• Impaired methylation contributes to DS risk
• Epigenetic regulation of gene expression
is the driver of development
•Genes on Chr21 can affect development via their
effect on cellular oxidative state and methylation status
• Metabolic support of methylation may help
optimize the abilities of DS individuals
Down Syndrome Trisomy Chr 21:
Trisomy 21 Causes Down Syndrome
By: Clare O'Connor, Ph.D. (Biology Department, Boston College) © 2008 Nature Education
Nondisjunction of linked sister chromatids can occur
at two different stages of egg (or sperm) development:
Meiosis I and Meiosis II
Trisomy begins with maternal gametogenesis, which
occurs within the maternal grandmother’s body,
during her pregnancy.
Prophase
Oocytes
form
during
maternal
fetal
development
Meiosis I
Meiosis II
Faulty
Gamete
Production
(mainly ova)
Ovulation
and
Conception
Years
Maternal
Grandmother
Mother
or
Father
Prenatal
Development
Mother
and
Placenta
Postnatal
Development
Breastfeeding
and
Nutrition
Methylation plays an important role in all
phases of conception and development
Epigenetic Changes: Methylation of DNA or Histone
DNA Methylation
DNA
Methyltransferase
Cytosine
5-Me Cytosine
Sites for Histone
Methylation
Regulation of gene expression during development
Transcription Factor Regulation:
Growth Factors
Neurotransmitters
Hormones
Start site for
mRNA synthesis
CpG TF CpG
TF binding region
DNA
Gene sequence
RNA
polymerase
SAM
Protein
(e.g. enzyme)
Me
MBDP
Me
HMT
Histone
proteins
(e.g. MeCP2)
DNA
Translation
X
Epigenetic Regulation:
DNMT
mRNA
Transcription
Me
Me
CpG
CpG
TF binding region
SAM
Me
Me
X No mRNA
DNA + Histone = Heterochromatin
Genes are silenced and transcription is blocked
METHIONINE CYCLE
Adenosine
A reversible reaction!
SAH
HCY
MethylTHF
Methionine
Synthase
Vitamin B12
(Cobalamin)
THF
SAM
MET
ATP
(-)
~1,000
Methylation
(+)
Reactions
via
209 MTases
PP + Pi
Global methylation =
DIETARY PROTEIN
( SAM
)
SAH
Glutathione (GSH)
Redox = GSH
Status GSSG
γ-Glutamylcysteine
Transsulfuration
Cysteine
Cystathionine
D4 Dopamine
Receptor
PLM Cycle
Adenosine
D4-SAH
Adenosine
CBS
D4-HCY
HCY
THF
D4-SAM
PP + Pi
SAH
(-)
Methyl-THF
Methyl-THF
Phospholipid
Methylation
Methionine
Cycle
Methionine
Synthase
D4-MET
ATP
Dopamine (Attention)
THF
> 1,000
Methylation
Reactions
SAM
MET
ATP PP + Pi
24% decrease in HCY in DS
47% decrease in MET in DS
350% increase in cystathionine
15% decrease in cysteine in DS
Consistent with a decrease in methionine
synthase activity and an increase in CBS activity
25% decrease in combined SAM and SAH in DS
33% increase in adenosine in DS
Consistent with a decrease in methionine
synthase activity and an increase in CBS activity
In DS:
Increased transsulfuration
Decreased methylation
Glutathione (GSH)
Redox = GSH
Status GSSG
γ-Glutamylcysteine
↑Transsulfuration
Cysteine
Cystathionine
D4 Dopamine
Receptor
PLM Cycle
Adenosine
Adenosine
CBS
↓Methionine
Cycle
D4-SAH
D4-HCY
HCY
THF
D4-SAM
PP + Pi
(-)
Methyl-THF
Methyl-THF
Phospholipid
Methylation
SAH
Methionine
Synthase
D4-MET
ATP
Dopamine (Attention)
THF
> 1,000
Methylation
Reactions
SAM
MET
ATP PP + Pi
Methionine synthase has five domains + cobalamin (Vitamin B12)
Domains alternate interacting with cobalamin during turnover
HCY Domain
SAM Domain
3
B12 serves as
a sensor of
redox status
2
1
Cobalamin
(vitamin B12)
Cobalamin
Domain
Cap
Domain
5-Methyl THF Domain
Cognitive
Status
Nitric Oxide
Synthesis
Catecholamine
Methylation
REDOX
STATUS:
GSH
GSSH
Methylation
Status:
SAM
SAH
Creatine
Synthesis
Arginine
Methylation
~ 1,000
Methylation
Reactions
Phospholipid
Methylation
Gene
Expression
DNA/Histone
Methylation
Serotonin
Methylation
Melatonin
Energy
Status
Membrane
Properties
Sleep
Domain-specific PCR analysis of MS mRNA
in postmortem frontal cortex
Dr. Christina Muratore - 2010
Research Fellow in Neurology
Harvard Medical School
3'
HCY
FOL
187 bp
Exon 19
COB
197 bp
20
188 bp
CAP
5'
SAM
419 bp
21
22
23
24
25
122 bp
Age-dependent decrease of Cob and Cap domain
mRNA in postmortem frontal cortex in 49 subjects
Cobalamin-binding Domain
Cap Domain
Methionine synthase mRNA is lower but protein levels
are not different in cortex of autistic subjects
Control
Autistic
C1 C2 C3 C4 C5
A1 A2 A3 A4 A5
MW(kD)
250
150
100
Methionine synthase provides redox-sensitive
global coordination of metabolism: HOMEOSTASIS
METHIONINE SYNTHASE
REGULATION
GLOBAL
REGULATION
DNA Methylation MS DNA
(- )
TNF-α
Ubiquitination
(- )
Amino
Acids
MS pre-RNA
Alternative
Splicing
MET
GSH
(- )
HCY
Translation
MET t-RNA
SAM
MS
Cysteine
Protein
Methylation
MS RNA
Cellular
Redox Status
(+ )
SAH
Cystathionine
[SAM]
[SAH]
Methylation
Global
Metabolic
Coordination
RNA
Splicing
DNA
Transcription
RISK OF DS
In a number of studies (but not all)
impaired methylation status and/or
elevated homocysteine in the
mother was shown to influence the
risk of Down Syndrome
(i.e. poor methylation may increase
the risk of nondisjunction)
But, what about grandma?
After 10 years (1999–2009) of active research in the field the
question of whether or not polymorphisms in folate/Hcy
metabolizing genes are associated with increased DS risk is still
largely debated in literature, and none of the studied
polymorphisms can be firmly considered as an independent DS
risk factor[15–40].
Even if MTHFR 677C > T, MTHFR 1298A > C and MTRR
66A > G gene polymorphisms gave positive results in several
independent studies, results are still conflicting and inconclusive
Increased HCY in mothers of DS individuals was found in a number of studies,
but was not found in other studies (e.g. in France). MTHFR 677C > T correlates
with increased HCY.
Genetic variants in methylation pathway associated with DS
CBS
(833 ins 68)
Combinations of methylation-related SNPs increase the maternal risk of
having a DS baby in some studies (e.g. up to a 7-fold increase).
Higher HCY levels are associated with lower IQ.
Zampieri et al. (2012) found
significant risk associations
for maternal age, MTHFR 677 C>T,
and Transcobalamin 766 C>G, but
decreased risk for BHMT 742 G>A.
DS children have a lower SAM/SAH, indicative of impaired methylation capacity
Role of oxidative stress and
methylation during development
Sperm are rich
in selenium
Eggs are rich
In cysteine
GSH
Does Redox Control
Development Via
Epigenetic Effects?
∆ Gene
Expression
GSH
GSH
∆ DNA and Histone
Methylation
GSH
GSH
GSH
juanv.wordpress.com/
Chromosome 21 Genes and Down Syndrome
Which chromosome 21 genes contribute to DS?
Answer: All of them contribute something.
Which genes are more important for causing Down Syndrome?
Answer: Methylation-related genes
Which genes are more important for cognitive development?
Answer: Methylation-related genes
• Chromosome 21 has about 400 genes. Increased gene dosage from
each of them probably contributes to Down Syndrome characteristics.
• Certain genes deserve special attention for their relationship to
oxidative stress and methylation, which are the foundation of
development, especially brain development.
• These genes is located in the region of chromosome 21 (21q21-22)
that has been implicated as being most important for DS.
• Increased activity of the proteins produced by these genes
is likely to contribute to DS.
Amyloid precursor protein (APP)
Cystathionine-beta-synthase (CBS)
DNA methyltransferase 3L (DNMT3L)
Formiminotransferase cyclodeaminase (FTCD)
Superoxide dismutase 1 (SOD1)
Formiminotransferase cyclodeaminase (FTCD):
Removes an imino group from forminoglutamate
(FIGLU), attaches it to THF, and converts it to
methenylTHF.
This provides a back-up source of methylTHF to
support methionine synthase and methylation.
In trisomy, a higher than normal activity of FTCD might provide extra
methyl groups to compensate for decreased MTHFR activity.
Formiminotransferase
cyclodeaminase
MTHFR
FIGLU
AMYLOID PRECURSOR PROTEIN (APP):
A cleavage product of amyloid precursor protein
(APP), known as Aβ, is though to be the primary
cause of Alzheimer’s disease (AD).
Amyloid plaques rich in Aβ are found at autopsy in
AD brain, but the neurodegeneration is thought to
be caused by small Aβ oligomers, starting decades
before the onset of AD symptoms.
AD is much more common in DS, presumably
because the extra APP gene leads to increased Aβ.
Current Genetics of Alzheimer’s Disease
• Early onset familial
AD
▫ APP
▫ Presenilin 1 and 2
• Late onset AD
▫
▫
▫
▫
▫
▫
▫
▫
▫
APOE4
LRP1
A2M
AD5-8
HLA-A
NOS3
PAXIP1
MS
MTHFR
OMIM.org
Amyloid Processing
http://www.bath.ac.uk/bio-sci/research/profiles/brown-d.html
Neurons have impaired transsulfuration and low GSH levels
that depend upon growth factor-stimulated cysteine uptake
Neurotrophic
Growth
Factors
Cysteine
Cysteinylglycine
GSH
Astrocytes
GSH
Cystine
EAAT3
PI3-kinase ( + )
GSSG
GSCbl
GSH
OHCbl
γ-Glutamylcysteine
EAAT3
SAM
MeCbl
Cysteine
PARTIALLY BLOCKED IN
NEURONAL CELLS
Cystathionine
Adenosine
D4SAH
Adenosine
D4HCY
MethylTHF
Phospholipid
Methylation
MethylTHF
Methionine
Synthase
THF
D4SAM
ATP
Dopamine
(-)
>1,000
Methylation
Reactions
THF
D4MET
PP+Pi
SAH
HCY
SAM
MET
ATP
PP+Pi
NEURON
7P
A2
-C
M
+
+
AW
7
7
AW
M
4
-C
M
-C
0
7P
A2
10
-C
M
20
SAM/SAH Ratio
30
CH
O
7
*
ro
l
AW
7
7P
A2
CM
+
+
7
AW
7
-C
M
AW
7P
A2
CM
CH
O-
Co
nt
ro
l
-C
M
CH
O-
8
CH
O
+
AW
50
Co
nt
2CM
+
0.0
7P
A
40
-C
M
2CM
L-[ 35S]-Cysteine Uptake
(nmol/mg protein)
*
CH
O
M
10
%
*
7P
A
0
-C
%
7PA2
CHO
CH
O
150
ro
l
50
GSH/GSSG Ratio
*
1%
0.
1
Co
nt
ro
l
L-[35S]-Cysteine
Uptake (nmol/mg protein)
0.5
nt
100
1.0
Co
Co
nt
ro
l
CH
O
-C
M
7P
A2
-C
CH
M
O
-C
M
+
7P
AW
A2
7
-C
M
+
AW
7
Intracellular Cysteine
nMol/mg protein
Soluble Aβ oligomers inhibit cysteine uptake,
increase oxidative stress and decrease methylation
capacity in neuroblastoma cells
1.5
1.5
1.0
*
0.5
0.0
6
*
2
0
A-β oligomers decrease DNA methylation
and alter expression of redox/methylation
pathway genes in neuroblastoma cells
0.5
*
*
#
0.3
0.2
*
*
*
*
0.1
on
tr
ol
C
H
O
-C
M
7P
A
2C
M
C
H
O
I
G
-C
FM
7P
1
7P
+
A
A
IG
22C
FC
M
M
1
+
+
IG
IG
FF1
1
+
W
M
N
LB
LB
TB
TB
A
A
C
+
H
IG
O
-C
F1
M
7P
+
A
A
2W
C
7
M
+
A
W
7
0.0
C
5-Methylcytosine (ng)
0.4
Our Amyloid Hypothesis
Abeta
Oligomers
Cysteine
Cysteinylglycine
GSH
Healthy
Glial Cells
(Astrocytes)
EAAT3
GSH
GSSG
(+)
γ-Glutamylcysteine
PI3-kinase
Cysteine
Cystine
Cystathionine
Epigenetic
Changes
Adenosine
Homocystine
MeCbl
Nate Hodgson
PhD Aug 2012
Methionine
Synthase
SAM
GSH
OHCbl
GSCbl
SAH
HCY
(-)
MethylTHF
Methyl-DNA
THF
DNA
SAM
MET
ATP PP+P
• A-β inhibits cysteine uptake, decreases
DNA methylation and alters gene expression.
• This is likely to be a natural role for A-β,
promoting oxidative stress in neurons.
• The extra APP and A-β production occurring in
trisomy 21 may produce excessive oxidative
stress, with adverse epigenetic consequences.
Superoxide dismutase 1 (SOD1)
• Superoxide anion is a reactive oxygen species (ROS)
produced by mitochondria as a by-product during
ATP synthesis
• SOD converts superoxide to hydrogen peroxide
Interestingly, methionine synthase has B12-dependent
SOD activity, which is essential for GSH-dependent
reactivation of enzyme activity after B12 oxidation
Data from Waly et al. (In Prep)
Cystathionine beta synthase (CBS):
CBS converts HCY to cystathionine in a vitamin B6dependent reaction
•
CBS activity is increased by SAM but increased by
oxidative stress and TNF-alpha
•
Vitamin D was recently shown to increase the level of
CBS
•
•
Testosterone decreases CBS activity
•
CBS also converts cysteine to hydrogen sulfide
Transsulfuration
Pathway
Glutathione (GSH)
SAM
Vitamin D
Oxidative stress
THF-alpha
(+)
Testosterone
Cysteine
Cystathionine
Adenosine
CBS
(-)
~ GSH
=
GSSG
γ-Glutamylcysteine
Cysteine
Hydrogen
Sulfide
Redox
Status
HCY
SAH
(-)
Methyl-THF
Methionine
Synthase
THF
> 1,000
Methylation
Reactions
SAM
MET
ATP PP + Pi
Table 2 | Intellectual disability by gender (n=121)
mild
moderate
severe
profound
Males
count
9
23
21
17
%
12.9
32.9
30.0
24.3
Females
count
%
14
27.5
13
25.5
19
37.3
5
9.8
Total
count
23
36
40
22
%
19.0
29.8
33.1
18.2
Males are affected more severely than females
Excessive CBS activity in DS limits
methylation by removing HCY from
the methionine cycle.
The critical balance between methylation
and transsulfuration is therefore altered.
DNA methyltransferase 3L (DNMT3L):
• Does not directly attach methyl groups to DNA,
but forms a complex with other methyltransferases
and alters their activity.
• Important for gender-specific DNA methylation
In the absence of Dnmt3L, neither methylation of most oocyte-methylated
gDMRs nor intragenic methylation was observed. There was also genomewide hypomethylation, and partial methylation at particular
retrotransposons, while maintaining global gene expression, in oocytes.
Along with the identification of the many Dnmt3L-dependent gDMRs at
intragenic regions, the present results suggest that oocyte methylation
can be divided into 2 types: Dnmt3L-dependent methylation, which is
required for maternal methylation imprinting, and Dnmt3L-independent
methylation, which might be essential for endogenous retroviral DNA
silencing. The present data provide entirely new perspectives on the
evaluation of epigenetic markers in germline cells.
In other words, DNMT3L is critical for oocyte methylation.
SUMMARY
• > 400 genes on Chr 21 contribute to Down syndrome.
• The relatively high survival rates for Chr 21 trisomy indicates
that the higher expression of genes creates an acceptable
alternative pathway for development.
• Impaired methylation, importantly involving altered DNA
methylation, is a primary factor in causing DS.
• Altered patterns of methylation continue to be important
throughout the lifespan.
• Metabolic interventions which address oxidative stress and
improve methylation capacity may be beneficial.
• Controlled clinical trials of these interventions are needed.
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
Mostafa Waly
Yahya Al-Farsi
Grant Support:
Autism Research Institute
SafeMinds
National Autism Association
Autism Speaks