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Dysregulated redox balance associated with
glutamate excitotoxicity in autism spectrum
disorders
Prof. Afaf El-Ansary
Department of Biochemistry
Science College, King Saud University
[email protected]
Background
 Autism
spectrum
disorders
(ASD) are characterized by three
core behavioral domains: social
deficits,
impaired
communication, and repetitive
behaviors.
 Glutamate
excitotoxicity has
been found in various preclinical
models of this disorder.
 Inefficient detoxification system
leads to oxidative stress, gut
dysbiosis,
and
immune
dysfunction has been also
accepted
as
etiological
mechanism of autism.
This work is an attempt to understand the
relationship between glutamate
excitotoxicity and impaired detoxification
as two mechanisms recently related to
autism.
 Central Nervous System (CNS)
• Brain & Spinal Cord
 Peripheral Nervous System (PNS)
• Afferent (sensory) Nerves –
Carry sensory information to
the CNS
• Efferent (motor) Nerves –
Transmit information to
muscles or glands
Cells of the Nervous System
 Neurons
• Signal integration/generation; direct
control of skeletal muscle (motor axons)
 Supporting Cells (Glia cells)
• Astrocytes (CNS – blood brain barrier)
• Oligodendrocytes (CNS – myelination)
• Schwann cells (PNS – myelination)
• Microglia (activated astrocytes)
Cellular Events in Neurodevelopment
Events:
 Division
 Migration
 Differentiation
 Neurogenesis
 Formation of synapses
 Myelination
 Apoptosis
Active
throughout
childhood &
adolescence
Why is the Brain Particularly
Vulnerable to Injury?
 Neurons are post-mitotic cells
 High dependence on oxygen
•
Little anaerobic capacity
•
Brief hypoxia/anoxia-neuron cell death
 Dependence on glucose
•
Sole energy source (no glycolysis)
•
High metabolic rate
 Many substances go directly to the brain via inhalation
Blood Supply to the Brain
Blood-brain Barrier
 The BBB consists of around 500 miles of blood
vessels throughout the brain, all packed with
endothelial cells that are highly selective about
what gets through to the brain. With few
exceptions, only fat-soluble small molecules
penetrate - alcohol, caffeine, and nicotine are
among them.
BBB can be broken down by
 Hypertension: high blood pressure opens the




BBB
Hyperosmolarity: high concentration of
solutes can open the BBB.
Infection: exposure to infectious agents can
open the BBB.
Trauma, Ischemia, Inflammation, Pressure:
injury to the brain can open the BBB.
Development: the BBB is not fully formed at
birth.
Glutamate Synapses
Excitatory synapse of
brain
Required to generate
action potentials
Both
AMPA
and
NMDA receptors are
critical for normal brain
function
NMDA-high
permeability
Ca++
Glutamate/glutamine cycle
 It is well known that glial cells,
mainly
astrocytes
surround
glutamatergic synapses, and
express glutamate transporters
and the glutamate-metabolizing
enzyme, glutamine synthetase
(GS).
 Glutamate is transported into
glial cells and amidated by GS to
the non-toxic amino acid
glutamine. Glutamine is then
released by glial cells and taken
up by neurons, where it is
hydrolyzed by glutaminase to
form glutamate again, completing
the glutamate/glutamine cycle.
 Appropriate clearance of
synaptic glutamate is
required for the normal
function
of
brain
excitatory synapses and
hence for prevention of
neurotoxicity recorded in
patients with autism.
Brain oxidative stress
 The
central nervous system
presents high vulnerability to free
radical damage due to its elevated
oxidative metabolic rate and
enriched content of unsaturated
lipids, as well as to its elevated
rate of free radical generation
derived from neurotransmitters
metabolism, and poor radical
scavenging mechanisms.
 It is also hypothesized that
autistic patients
as poor
detoxifiers have reduced ability
to eliminate mercury. Higher
levels of mercury were recently
found to be associated with
social impairment and severity of
autism.
ANTROPOGENIC
EMISSIONS
NATURAL EMISSIONS
oVolcanoes
oRock erosion
oFires
oCoal power
plants
oCement
production
Methylation
Plankton
OTHER POTENTIAL
SOURCES
oFolk medicines
oCosmetics
oAmalgams
oVaccines
Excitotoxicity-Glutamate Mediated Cell Death
Glutamate
induces a delayed cell death in neurons
This
cell death requires extracellular calcium and is
blocked by antagonists of NMDA receptors
Action
potentials are initiated in the nerve axon after
glutamate excitatory activation of receptors in the
neuron's dendrites and cell body.
Hypothesis:
Prolonged or inappropriate activation of
NMDA receptors underlies glutamate excitotoxicity of
neurons .
Methodology
20 male patients with autism age of 8 ± 4 were
enrolled through the Autism Research and
Treatment (ART) Center clinic. The diagnosis of
ASD was confirmed in all subjects using the
Autism Diagnostic Interview-Revised (ADI-R)
criteria, the Autism Diagnostic Observation
Schedule-Generic (ADOS-G) criteria and the
Developmental, Dimensional and Diagnostic
Interview (3DI). Another age and sex matching 30
as a control group.
Samples collection
After overnight fast, 10 ml blood samples were
collected from autistic and control groups in test
tubes containing anticoagulant. Tubes were
centrifuged at 1000 rpm at room temperature for 15
minutes, plasma was obtained and deep freezed (at
-80°C) and RBCs were kept at -20°C until
analysis time.
Biochemical assays:
Glutamate, glutamine, thioredoxins I&III,
Thioredoxin
reductase,
peroxidoxins,
glutathione status and glutathione-stransferase were measured in plasma
samples of autistic children compared to
controls using ELISA kits. Mercury was
measured using atomic absorption.
1.
Independent t-test
• Independent t-test to compare between the 2 studied groups.
• Results were expressed as means ± S.D. Statistical comparisons
were performed with independent t-tests using (SPSS).
• Significance were assigned at the level of P < 0.05.
2. ROC analysis
Area under the curve, cutoff values, and degree of specificity
and sensitivity were calculated.
Table 1: Table 1 Mean ± SD of the measured chemicals in plasma or red blood cells of patients with autism compared
with age-matched controls
Parameter
Group
N
Mean ± S.D.
Percent Change
Control
20
111.91 ± 4.51
100.00
Autistic
20
152.80 ± 6.47
136.54
Control
20
241.82 ± 12.93
100.00
Autistic
20
111.34 ± 5.69
46.04
Control
20
0.46 ± 0.03
100.00
Autistic
20
1.37 ± 0.06
296.18
Glutamate Dehydrogenase Control
(GLDH) (U/l)
Autistic
20
1.71 ± 0.47
100.00
20
0.93 ± 0.36
54.22
Control
20
44.71 ± 7.43
100.00
Autistic
20
74.70 ± 9.04
167.09
Control
20
1.83 ± 0.52
100.00
Autistic
20
3.31 ± 1.11
180.87
Control
20
19.58 ± 4.76
100.00
Autistic
20
34.56 ± 8.32
176.55
Control
20
24.30 ± 2.69
100.00
Autistic
20
43.05 ± 5.86
177.16
Control
20
26.07 ± 5.03
100.00
Autistic
20
8.03 ± 2.46
30.79
Glutathione S Transferase Control
activity (µmol/min/ml)
Autistic
20
0.69 ± 0.20
100.00
20
0.41 ± 0.12
59.26
Control
20
4.64 ± 0.68
100.00
Autistic
20
6.93 ± 0.74
149.40
Glutamic (µmol/l)
Glutamine (µmol/l)
Glutamic / Glutamine Ratio
Thioredoxin 1 (ng/ml)
Thioredoxin reductase
(mU/ml)
Peroxiredoxin 1 (ng/ml)
Peroxiredoxin 3 (ng/ml)
GSH /GSSG
Mercury (µg/L)
P value
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
Higher Hg can be easily
related to of GSH/GSSG status.
It is well known that sulfhydrylcontaining enzymes are inhibited
by MeHg.
With particular
toxicity induced
known that its
directly interacts
group of GSH,
formation of an
HgCH3 complex
emphasis on the
by MeHg, it is
mercury atom
with the thiol
leading to the
execretable GS-
A mechanism links dysregulated redox status
to glutamate excitotoxicity could be easily
proposed.
Simply, in the brain, ion gradients across
neural membranes is important for resting
membrane and this usually maintained by
ATP-dependent ion pumps, such as a Na+/K+
pump.
ATP depletion induces impairment in the
repolarization of neural membranes after a
depolarizing stimulus.
Defective repolarization can relieve a
voltage dependent Mg+ block of NMDA
channels, leading to persistent receptor
activation by endogenous glutamate.
This hypothesis is derived from an in vitro
experiment showing that the inhibition of
energy metabolism makes glutamate
neurotoxic at concentrations that ordinarily
show no toxicity.
Calcium entry via NMDA receptors can
trigger neuronal cell death
Table 2: ROC-Curve of all parameters in autistic groups
AUC
Best
Cutoff
value
Sensitivi
ty %
Specifici
ty %
1.000
131.110
100.0 %
100.0 %
1.000
169.800
100.0 %
100.0 %
Glutamic /
Glutamine Ratio
1.000
0.906
100.0 %
100.0 %
Glutamate
Dehydrogenase
(GLDH) (U/l)
0.915
1.575
100.0 %
75.0 %
Thioredoxin 1
level (ng/ml)
0.993
57.400
100.0 %
95.0 %
Thioredoxinredu
ctase activity
(mU/ml)
0.894
2.000
80.0 %
75.0 %
0.977
27.250
95.0 %
100.0 %
1.000
33.000
100.0 %
100.0 %
GSH / GSSG
1.000
16.340
100.0 %
100.0 %
Glutathione S
Transferase
activity
(µmol/min/ml)
0.900
0.505
85.0 %
95.0 %
Mercury (µg/L)
0.988
5.997
95.0 %
100.0 %
Glutamic
(µmol/l)
Glutamine
(µmol/l)
Peroxiredoxin 1
level (ng/ml)
Peroxiredoxin 3
level (ng/ml)
Table 3: Multiple Regression using Stepwise method for Glutamic (µmol/l)as
a dependent variable
Predictor Variable
Beta
P value
Glutamic (µmol/l)
0.006
0.001
Glutamine (µmol/l)
-0.005
0.001
Thioredoxin reductase
activity (mU/ml)
0.013
0.038
GSH / GSSG
-0.003
0.018
Model
Adjusted
R2
F value
P value
0.995
1938.823
0.001
Table 4: Multiple Regression using Stepwise method for Glutamine (µmol/l)as a
dependent variable
Predictor Variable
Glutamine (µmol/l)
Glutamic / Glutamine
Ratio
Peroxiredoxin 1 level
(ng/ml)
Beta
P value
0.302
0.001
92.604
0.001
-0.273
0.011
Model
Adjusted
R2
F value
P value
0.964
352.514
0.001
Table 5: Multiple Regression using Stepwise method for Glutamic / Glutamine
Ratioas a dependent variable
Predictor Variable
Glutamic (µmol/l)
Glutamic / Glutamine
Ratio
Thioredoxin 1 level
(ng/ml)
Beta
P value
0.969
0.001
-177.627
0.001
-0.274
0.044
Model
Adjusted
R2 square
0.990
F value
P value
1243.754
0.001
Based
on
this,
the
excitotoxicity suggested in the
present study as etiological
factor in autism can be related
to the impaired Prx I and III,
Trx,
Trxreductase
,
GSH/GSSG, and GST as
markers
of
impaired
detoxification mechanisms.
This could find support in the
study of Leveille et al
(2009)who
reported
that
excitotoxic insults can render
the neurons more vulnerable to
peroxidoxin
hyperoxidation
through the oxidation of cystine
to cysteine.
AUTISM
CONCLUSION
Mercury as neurotoxic metal could inhibit glutamate
transporters resulting in raising the levels of extracellular
glutamate by inhibiting antioxidant enzymes and
glutathione.
Mercury aggravates free radical generation and lipid
peroxidation.
Majority of children with autism exhibited high Hg,
mitochondrial dysfunction, and oxidative stress,
conditions that magnify glutamate excitotoxicity.
 This should call attention to glutamate signaling being
a major mechanism in damage to the autistic brain.
Stimulation of glutamate transporters could be a good
strategy to treat this disorder.