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MITOCHONDRIAL FUNCTIONS ABNORMALITY IN IDIOPATHIC AUTISM SPECTRUM DISORDER
17
OXIDATIVE STRESS AND MITOCHONDRIAL DYSREGULATION IN IDIOPATHIC ASD
DR. NORWAHIDAH A.K
UKM MEDICAL CENTRE
norwahidah@ppukm.ukm.edu.my
restricted, repetitive patterns of behaviour, interest and activity.
individual variation in the type and intensity of symptoms
deficits in social interaction and communication
neurodevelopmental disorders:
Autism Spectrum Disorder
(ASD)
Boys are four times more likely to develop autism than girls.
several cases associated with mental retardation
increasing prevalence
- The global prevalence of ASD has increased twenty-fold to thirty-fold since the earliest epidemiologic studies were conducted (late 1960s and early 1970s).
- At that time, prevalence estimates from European studies were one in 2,500 children in population.
- WHO:
- In 2019, one in 160 children worldwide
- Autism Speaks:
- In 2020, one in 54 children
- Malaysia, Ministry of Health:
- Current report, 300, 000 people with ASD
- economic impact :
- $268 billion for 2015
- $1 trillion for 2025
- ASD related medical, non-medical and productivity loses
UC Davis Health System,News Room
Topic 2
Symptoms
"Individuals with autism may exhibit:
Behavioural Challenges in ASD
- hyperactivity
- short attention span
- impulsivity
- aggression
- self-injurious behaviours
- odd responses to sensory stimuli
- eating, sleeping, motor or mood abnormalities "
(McGahan, 2001)
Frustration
Sensory Overload
Anxiety
FACTORS
UNPROVEN DATA
Mitochondria
- Pathogenesis remain elusive
- Variety factors have been implicated
- Controversial environmental factors:
- exposure to heavy metals
- immunization
- Genetics
- Immunological - activation of immune system in CSF and in the brain with neuroglia and inflammatory response
- Metabolic -dysfunction of glutamate and GABA neurotransmitters, abnormal function serotonin and dopamine
- MITOCHONDRIA ???
Mitochondrion
- intracellular organelle
- crucial role in ATP production
- through oxidative phosphorylation (OXPHOS)
- electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions.
ELECTRON TRANSPORT SYSTEM
ATP production
Complex I II III IV ATP synthase
ROS generation
- Antioxidant enzymes
- ROS/Oxidant
Research Breakthrough
Oxidative Stress, Differential Mitochondrial Bioenergetics and Atypical Morphology in ASD
Mitochondrial Bioenergetic,
Oxidative Stress
and Morphological Analysis
Mitochondrial Analysis in Autism Lymphoblastoid Cell Line
Mitochondrial Bioenergetics Analysis
High-Resolution Respirometry & Fluorometry
(Oxygraph-2K)
Elisa
Mitochondrial respiration
Definitions of substrates states and Coupling states
Elucidation of Mitochondrial Respiration
Routine (R): respiration regulated according to physiological activity, at non-saturating ADP level
Leak (L): respiration in the presence of reducing substrate but absence of ADP
OXPHOS (P): respiratory capacity in the ADP-activated state, inorganic phosphate, O2, and reduced substrates
CI-linked: -linked substrate state, induced by addition of NADH-generating substrates
CII-linked: -linked substrate state, induced by addition of succinate and rotenone (CI inhibitor)
CI&CII-linked: induced by addition of NADH-generating substrates in combination with succinate
ETS: capacity is max O2 flux at optimum uncoupler concentration
* denotes p<0.05 compared to NALCL
C II-Linked Respiration and mtMP by Safranin
Principles:
Safranin:
- fluorescent probes
- hydrophobic cations
- accumulates in mitochondria
- in response to negative potential generation
- in the energized state
- substrate is added to the system
- the fluorescence intensity drops in response to energization
- due to accumulation and quenching of fluorescence of the dye in the mito matrix.
Assessment of Complex II-Linked Respiration & Mitochondrial Membrane Potential
by Safranin
* denotes p<0.05 compared to NALCL
Safranin Signals
mtMP
* denotes p<0.05 compared to NALCL
Complex IV activity
Cells
Digitonin
Ama
AS TMPD
C
Azd
CCCP
Principle:
- Digitonin: permeabilizes the plasma membrane selectively
- Antimycin A (Ama) : inhibits complex III
- CCCP: protonophore, chemical uncoupler of OXPHOS
- Ascorbate (As): maintaining the TMPD in a reduced state
- TMPD: artificial substrate for reducing cyto C
- Cyto C: transfers electrons from complex III to Complex IV
- Azd: inhibits complex IV
Determination of Complex IV activity
* denotes p<0.05 compared to NALCL
* denotes p<0.05 compared to NALCL
*
Complex V ATP Synthase
Activity
Higher respiratory capacity and mtMP in ALCL
- The higher ROUTINE respiration rate in ALCL - a compensatory response to the intrinsic energy demand.
- Increased respiration rate leads to higher oxidative stress and alterations in mitochondrial energy metabolism.
- Higher OXPHOS- and ET-capacities would result in increased production of H2O2 and other ROS inducing oxidative stress - frequently observed in ASD patients compared to controls.
- Our results are in line with another study that reported a 40-50% increase in maximal respiratory rates in lymphoblasts of ASD patients as compared to the non-autism relative.
Reports
Chauhan et al. 2011, Giulivi et al. 2010, Benzecry et al. 2008
- mtMP reflects the electrical potential difference across the mitochondrial inner membrane, which in conjunction with the driving force for ATP synthesis.
- In our study, the mtMP in autism was higher in CII-OXPHOS respiration as compared to normal which in agreement with previous studies in ASD patients.
- The higher the mtMP would reflect the higher the energy capacity of the mitochondrial inner membrane and the higher the synthesis of ATP.
- Mitochondrial ROS production by mitochondrial transport system is increased at high membrane potential.
- Thus, high mtMP is potentially harmful to mitochondria and consequently to the cell.
Chauhan et al. 2009, James et al. 2009, Zorova et al. 2018, Suski et al. 2012, Korshunov et al. 1997, Skulachev et al. 1996
Overactivities of C IV and C V
- CIV is the terminal respiratory complex of the electron transfer system, which converts O2 to H2O which is used by CV.
- The generated proton gradient is used by CV (ATP synthase) to catalyze the formation of ATP by the phosphorylation of adenosine diphosphate (ADP).
- Increased activity of CIV was detected in brain tissue and complex V in muscle tissue of ASD.
- In line with our study, the CIV and V activities were higher in autism compared to normal.
- This occurs as to cope with the increase in the ET-capacity of CI and CII,
- transfer the electrons towards CIV
- increased in complex V activity
- to catalyze the formation of ATP.
Chauhan et al. 2012, Rossignol & Frye 2012, Oliveira et al 2006, Filiano 2002, Palmieri et al. 2010, Frye et al. 2011
Oxidative Stress and DNA Damage
Oxidative Stress Analyis
(ELISA)
* denotes p<0.05 compared to NALCL
*
OXIDATIVE DAMAGES
*
a : denotes p<0.05 compared to NALCL
*
- Studies have found that individual with ASD display hallmarks of
- increased oxidative stress or
- abnormalities in redox regulation
- suggesting the mechanistic role of ROS in the patho ASD
- SOD, CAT, and GPx play a vital role in protecting cells from oxidative stress
- to halt the ROS production and deactivate the propagated ROS
- oxidative stress occurs when ROS overpowers the antioxidant defense mech
- oxidative damages
- A higher level of ROS was generated in ASD children,
- indicated by the decrease of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in erythrocytes
Griffithf & Levy 2017, Manivasagam et al. 2020, Sogut et al. 2003
Normal physiology
- Complexes I and III are the main sites of mitochondrial superoxide production
The ETC itself is the target of ROS, leads to
- ETC complexes impairment
- inhibit ATP synthesis
- accelerate ROS generation
- energy metabolism impairment,
- oxidative stress, disruption of mitochondrial functions,
- affect neurons’ function and plasticity,
- neurodevelopmental disorders in children.
(Chauhan et al. 2012)
High mtMp subsequently increased the mitochondrial ROS production.
(Markkanen et al. 2016)
- Evidence of increased oxidative damage to DNA, proteins and lipids
has been identified in blood, urine, and post-mortem brain samples from ASD patients. (Rossignol & Frye 2014, Griffithf & Levy 2017)
- The incidence of DNA oxidation damage and a deficit in antioxidant capacity in the plasma of ASD children compared to non-affected siblings.
Our data disclosed a significantly more severe DNA damage of Grade 2 and 3, indicating higher levels of DNA damage occurring in the autism cells than the normal cells. These results were congruent with the previous reports.
These studies suggested that increased oxidative stress in ASD may originate from mt function abnormality.
Morphological Analysis
Normal
intact typical rod shape and smooth surface
ALCL (autism)
distorted shape
presence of blebbing and punched out lesions
- Some studies described specific ultrastructural abnormalities, such as
- abnormal cristae
- inclusions
- abnormally shaped and sized mitochondria
in children with ASD
- Animal models of ASD with abnormal function of the energy pathway also showed abnormal morphology of mitochondria
Rossignol & Frye 2012
The deformities we observed on the OMM may reflect direct damage of the mitochondria due to susceptibility of ALCL mitochondria towards oxidative stress.
Our findings may hypothesised,
- dysregulation in ETS- significant higher in mt respiration, mtMP, complex IV and V may further lead to
- accelerate ROS generation
- compromised endogenous antioxidant defense mechanism
- oxidative stress
- oxidative damages
- disrupt mitochondrial functions
- pathogenesis of ASD - neuronal damage
ASD PATHOGENESIS
ROS
Complex IV
ROS
CONCLUSION
Mitochondrial morphology abnormality
Complex V
mtMP
DNA Damage
??????????
Mt Copy number
Mt Density
Mutations
Gene/Protein expression
Mitochondrial respiration
SOD
Ongoing studies:
- Hydrogen Proxide production
- Proteomic & Genomic Study:
1. MtDNA sequencing using NGS
2. Microarray analysis
- Intervention using antioxidants:
1. Tocotrienol-Rich Fractions
2. Stingless Bee Honey (Kelulut)