In the present investigation, we demonstrate for the first time that the decreased glutathione-mediated redox/antioxidant capacity previously observed in plasma and immune cells from children with autism is also significantly decreased in two brain regions previously shown to be affected in autism, the cerebellum and BA22. Our findings also confirm previous preliminary reports that markers of oxidative damage (3-NT and 8-oxo-dG) are increased in these two brain regions in individuals with autism.32, 44
We further extend these findings by examining a larger sample of carefully selected tissues for multiple markers of oxidative protein/DNA damage (3-NT, 8-oxo-dG), as well as functional biomarkers of inflammation (3-CT) and mitochondrial superoxide production (aconitase activity).
A relative decrease in aconitase activity in the autism cerebellum is an important new finding, suggesting a functional consequence of oxidative stress in this region. In brain tissue, aconitase is located primarily in the mitochondria where it functions as an enzyme in the tricarboxylic acid cycle.37
Mitochondrial aconitase is highly sensitive to oxidative inactivation by superoxide radicals that are produced in close proximity by the electron transport chain (ETC). Thus, in addition to being a marker of oxidative protein damage, a decrease in aconitase activity is considered to be a sensitive indicator of elevated mitochondrial superoxide production.45, 46
The labile iron–sulfur (Fe-S) cluster present in the active site of aconitase is a major target of excessive mitochondrial superoxide. In the presence of sufficient reducing agents, such as GSH or NADPH, aconitase can be restored to its active form;47
however, in the autism cerebellum, the observed decrease in GSH concentration relative to controls indicates a chronic deficit of reducing equivalents in this region. As depicted in , unscavenged superoxide inactivates aconitase by displacing Fe+2
from the Fe-S cluster, which then promotes the formation of the damaging hydroxyl radical via reaction with H2
and Fenton chemistry. A fragile redox state within the mitochondria of individuals with autism has been previously reported,48
and may reflect a self-amplifying cycle of antioxidant depletion and aconitase inactivation. Several enzymes involved in ATP production contain Fe-S clusters in the active site and are subject to similar inactivation by superoxide, including oxoglutarate dehydrogenase of the tricarboxylic acid cycle, as well as ETC complexes I–III.49
A deficit in the tricarboxylic acid cycle and ETC function under conditions of excessive superoxide production in the brain would be expected to result in a reduced ability to maintain adequate levels of ATP required for normal neuronal and synaptic functioning.
Figure 4 Mechanism of mitochondrial aconitase inactivation. Mitochondrial aconitase is a tricarboxylic acid (TCA) cycle enzyme that catalyzes the conversion of citrate to isocitrate. It contains an iron-sulfur cluster ([4Fe-4S]) in its active site that is inactivated (more ...)
Aconitase inactivation and oxidative stress have been noted in other neuropsychiatric and neurodegenerative disorders with known mitochondrial involvement, including schizophrenia,50, 51
and Parkinson's disease.53, 54
One study of mitochondrial dysfunction in the autism brain found decreased protein levels of multiple ETC complexes in the cerebellum, frontal and temporal cortex.34
There is mounting evidence that mitochondrial dysfunction may be present in a significant subset of children with autism and contribute to the multisystem abnormalities seen in some autistic children.55, 56, 57
The significant decrease in aconitase activity warrants continued investigation into interactions between mitochondrial dysfunction, superoxide production and altered ETC complex activity in autism.
The oxidized protein tyrosine derivative, 3-NT, provides a stable biochemical footprint of oxidative protein damage and has been found to be elevated in plasma of children with autism in a previous study.31
Elevated levels of 3-NT have been described in a number of diseases with an oxidative stress pathology, including alcoholism, smoking, diabetes, atherosclerosis and cystic fibrosis.58
The tyrosine derivative, 3-NT, is formed primarily from peroxynitrite, a damaging free radical generated from superoxide and NO. Thus, the significant increase in levels of 3-NT observed in the autism cerebellum and BA22 was not unexpected and is consistent with elevated superoxide production and aconitase inactivation.
In addition to being a classic marker of oxidative protein damage, elevated levels of 3-NT indicates elevated NO production. Excessive NO competes with the antioxidants, MnSOD and GSH, for superoxide and promotes the generation of peroxynitrite.59
NO can reversibly inhibit mitochondrial respiration at complex IV, whereas the more damaging peroxynitrite can permanently inactivate complexes I, III and V.60
In a previous study, we demonstrated the increased sensitivity of autism lymphoblastoid cells to acute NO-induced mitochondrial membrane depolarization, and others have reported elevated plasma and red blood cells levels of nitrites in children with autism.24, 48, 61, 62
Neuroglial cells express iNOS (inducible NO synthase) and produce high quantities of NO when activated by cytokines.63, 64
Interestingly, the presence of proinflammatory cytokines and activated neuroglia have been reported in the autism cerebellum among other regions,9
suggesting that activated neuroglia produce excess NO and may contribute to the peroxynitrite formation and increased 3-NT protein damage observed in the present study.
A significantly elevated level of 3-CT in the autism cerebellum and BA22 is a novel finding indicative of a chronic neuroinflammatory state in these regions. Activated phagocytic cells produce hypochlorous acid, the product of myeloperoxidase (MPO) activity that is stimulated during immune activation, resulting in the 3-CT derivative.35
Elevated expression of MPO has previously been demonstrated in chronic neurological disease states, such as Alzheimer's disease,65
and multiple sclerosis.67
The observed increase in 3-CT in the autism cerebellum and BA22 samples is the first indication of elevated MPO expression in the autism brain, and supports previous reports of microglial activation and inflammatory cytokines in autism cerebellum.9
The role of inflammation and microglial activation in the neuropathology of autism warrants further investigation and confirmation.
In addition to markers of protein oxidative damage, 8-oxo-dG, a marker of DNA oxidative damage, was significantly elevated in both cerebellum and BA22 from the autism cases relative to controls. The 8-oxo-dG adduct in the mitochondrial and nuclear DNA is a pre-mutagenic lesion formed primarily by an attack by the hydroxyl radical (•
OH). It has been associated with oxidative DNA damage in conditions such as aging, cancer and pro-oxidant environmental exposures.68
In the cerebellum, 8-oxo-dG was negatively associated with GSH/GSSG in the combined case and control cohort (); however, this association failed to reach significance in the BA22 region. The superoxide-mediated release of Fe+2
associated with mitochondrial aconitase inactivation has been shown to be a significant source of OH radical formation through Fenton chemistry.47
Taken together, these data suggest the reduced GSH/GSSG antioxidant capacity is insufficient to counter excessive •
OH production, and that unopposed •
OH can reach the nucleus to create the oxidative DNA adduct, 8-oxo-dG.
The hypothesized interactions between each of the measured biomarkers of oxidative stress and damage, mitochondrial dysfunction and inflammation are diagrammed in . Elevated superoxide generated from dysfunctional mitochondria promotes the formation of excessive H2O2, the substrate for MPO-mediated hypochlorous acid synthesis and the generation of the inflammatory biomarker, 3-CT. Elevated superoxide can combine with NO, resulting in the formation of the peroxynitrite radical and the protein oxidative damage biomarker, 3-NT. The hydroxyl radical is generated by both aconitase inactivation and MPO activity, and promotes the formation of 8-oxo-dG. Chronic elevation of these free radicals will deplete GSH/GSSG redox/antioxidant capacity, allowing unopposed free-radical generation and a self-perpetuating cycle, leading to chronic oxidative stress and damage. The lack of a correlation between age and the biomarkers suggests that oxidative stress is a chronic condition in autism, because the same pattern of elevated biomarkers is seen over such a wide age range.
Figure 5 Proposed interactions between measured biomarkers and oxidative stress. Elevated superoxide generated from dysfunctional mitochondria promotes the formation of excess H2O2, the substrate for myeloperoxidase (MPO)-mediated hypochlorous acid (HOCl) synthesis (more ...)
In summary, we show for the first time that peripheral markers of oxidative stress and damage previously observed in plasma and immune cells are similarly elevated in two affected brain regions in autism, cerebellum and BA22. Together, these observations suggest that a pro-oxidant environment and oxidative stress are pervasive and systemic in individuals with autism. The negative association between GSH/GSSG, and oxidative protein and DNA damage suggest that decreased glutathione redox capacity in the autism brain may have functional consequence in terms of increased mitochondrial superoxide production and a chronic inflammatory state. Nonetheless, because autism is influenced by multiple interacting genetic and environmental factors that are case-specific and inherent limitations in post-mortem brain, these observations will require confirmation in subsequent studies.