Utilizing treated wastewater in tree plantation in Indian desert: part I - species suitability, plant growth and biomass production.

Treated wastewater can partly meet the requirements of water for irrigating tree crops in dry areas to better utilize, produce biomass and reduce land degradation. Seedlings of Acacia nilotica, Azadirachta indica, Eucalyptus camaldulensis, Prosopis cineraria, P. juliflora, Tamarix aphylla, Salvadora persica, S. oleoides and Tecomella undulata were planted and irrigated with bore-well (BW) and treated wastewater (WW) at ½ET (Evaporation-transpiration) and ¾ET. Plants irrigated at BW1/2 attained less height and collar diameter, and showed low growth increments and dry biomass. These variables increased by 1.2-2.0-fold at WW3/4 irrigation. Plants produced 1.4-fold higher biomass with WW over BW, whereas it was 1.5-fold higher at ¾ET over ½ET. Responses of species to BW/WW irrigation differed in biomass allocation to roots and other parts as a mechanism to uptake water and nutrients. Conclusively, A. nilotica, A. indica, P. juliflora and T. aphylla had stronger preference to increased irrigation level, whereas E. camaldulensis, S. persica, S. oleoides and T. undulata had preferred nutrients added through treated wastewater. A. nilotica, E. camaldulensis, P. juliflora and A. indica were best species in growth and biomass production and wastewater utilization. These can be replicated in urban afforestation to enhance bio-product and reduce degradation in environmental quality.

Wastewater (WW) is an alternative source of water in dryland afforestation. Wastewater was characterized and utilized in growing tree plantation in Indian arid zone. Plant growth and biomass increased with treated wastewater irrigation. Responses of different species to wastewater differed depending on their phytoremediation ability by nutrient uptake and biomass distribution. Tree species differed in their preferences to increased quantity of irrigation water and nutrients added through wastewater application.

Effects of Starvation on the Levels of Triglycerides, Diacylglycerol, and Activity of Lipase in Male and Female Drosophila Melanogaster.

We studied the effects of starvation on changes in neutral lipids in male and female Drosophila melanogaster (fruit fly) at different ages. When flies were subjected to starvation, the mortality rate was observed to be age- and gender-dependent: male flies died earlier as compared to female flies, and older flies died earlier than younger flies. There was an increase in the number of dead flies and the levels of diacylglycerol (DG) with starvation time. This increase in DG was observed much earlier in male flies as compared to female flies, which correlated with earlier death in male flies during starvation in comparison to female flies. We also analyzed the levels of triglycerides (TG) and lipase activity during starvation of flies. The levels of TG decreased depending upon the duration of starvation in both male and female flies. Interestingly, we observed that like DG, there was also an increase in lipase activity due to starvation, which also correlated with earlier death in male flies as compared to female flies. Our results suggest that increase in DG levels and lipase activity due to starvation may be the main cause of death in the flies.

Beneficial Effects of Walnuts on Cognition and Brain Health.

Oxidative stress and neuroinflammation have important roles in the aging process, mild cognitive impairment (MCI), Alzheimer's disease (AD), and other brain disorders. Amyloid beta protein (Aß) is the main component of amyloid plaques in the brains of people with AD. Several studies suggest that Aß increases the generation of free radicals in neurons, which leads to oxidative damage and cell death. Aß can also induce neuroinflammation by increasing pro-inflammatory cytokines and enzymes. Walnuts contain several components that have antioxidant and anti-inflammatory effects. Animal and human studies from our and other groups suggest that supplementation with walnuts in the diet may improve cognition and reduce the risk and/or progression of MCI and AD. In the transgenic AD mouse model (AD-tg), we have reported the beneficial effects of a diet with walnuts on memory, learning, motor coordination, anxiety, and locomotor activity. Human clinical trials have also suggested an association of walnut consumption with better cognitive performance and improvement in memory when compared to baseline in adults. Our recent study in AD-tg mice has shown that a walnut-enriched diet significantly improves antioxidant defense and decreases free radicals' levels, lipid peroxidation, and protein oxidation when compared to a control diet without walnuts. These findings suggest that a diet with walnuts can reduce oxidative stress by decreasing the generation of free radicals and by boosting antioxidant defense, thus resulting in decreased oxidative damage to lipids and proteins. An in vitro study with synthetic Aß showed that walnut extract can inhibit Aß fibrillization and solubilize the preformed Aß fibrils, suggesting an anti-amyloidogenic property of walnuts. Because it takes many years for cognitive impairment and dementia to develop, we suggest that early and long-term dietary supplementation with walnuts may help to maintain cognitive functions and may reduce the risk of developing, or delay the onset and/or slow the progression of, MCI and dementia by decreasing Aß fibrillization, reducing oxidative damage, increasing antioxidant defense, and decreasing neuroinflammation. Furthermore, several animal and human studies have suggested that walnuts may also decrease the risk or progression of other brain disorders such as Parkinson's disease, stroke, and depression, as well as of cardiovascular disease and type 2 diabetes. Together, these reports suggest the benefits of a walnut-enriched diet in brain disorders and in other chronic diseases, due to the additive or synergistic effects of walnut components for protection against oxidative stress and inflammation in these diseases.

Traumatic injury in female Drosophila melanogaster affects the development and induces behavioral abnormalities in the offspring.

Traumatic injury (TI) during pregnancy increases the risk for developing neurological disorders in the infants. These disorders are a major concern for the well-being of children born after TI during pregnancy. TI during pregnancy may result in preterm labor and delivery, abruptio placentae, and/or fetomaternal hemorrhage. Drosophila melanogaster (fruit fly) is a widely used model to study brain and behavioral disorders in humans. In this study, we analyzed the effects of TI to female fruit flies on the development timing of larvae, social interaction and the behavior of offspring flies. TI to the female flies was found to affect the development of larvae and the behavior of offspring flies. There was a significant increase in the length of larvae delivered by traumatically injured maternal flies as compared to larvae from control maternal flies (without TI). The pupae formation from larvae, and the metamorphosis of pupae to the first generation of flies were faster in the TI group than the control group. Negative geotaxis and distance of the fly to its nearest neighbor are parameters of behavioral assessment in fruit flies. Negative geotaxis significantly decreased in the first generation of both male (p = 0.0021) and female (p = 0.0426) flies. The distance between the first generation of flies to its nearest neighbor was shorter in both male and female offspring flies in the TI group as compared to control group flies. These results indicate that TI to the female flies affected the development of larvae and resulted in early delivery, impaired social interaction and behavioral alterations in the offspring.

Walnut Supplementation in the Diet Reduces Oxidative Damage and Improves Antioxidant Status in Transgenic Mouse Model of Alzheimer's Disease.

Our previous study has shown beneficial effects of walnuts on memory and learning skills in transgenic mouse model of Alzheimer's disease (AD-tg). To understand underlying mechanism, we studied here whether walnuts can reduce oxidative stress in AD. From 4 months of age, experimental AD-tg mice were fed diets containing 6% (T6) or 9% walnuts (T9) (equivalent to 1 or 1.5 oz, of walnuts per day in humans) for 5, 10, or 15 months. The control groups, i.e., AD-tg (T0) and wild-type (Wt) mice, were fed diets without walnuts. Free radicals, i.e., reactive oxygen species (ROS), lipid peroxidation, protein oxidation, and antioxidant enzymes were assessed in these mice at different ages. AD-tg mice on control diet (T0) showed significant age-dependent increase in ROS levels, lipid peroxidation, and protein oxidation coupled with impaired activities of antioxidant enzymes [superoxide dismutase, catalase, and glutathione peroxidase] compared to Wt mice. Oxidative stress was significantly reduced in AD-tg mice on diets with walnuts (T6, T9), as evidenced by decreased levels of ROS, lipid peroxidation, and protein oxidation, as well as by enhanced activities of antioxidant enzymes compared to T0 mice. Long-term supplementation with walnuts for 10 or 15 months was more effective in reducing oxidative stress in AD-tg mice. Our findings indicate that walnuts can reduce oxidative stress, not only by scavenging free radicals, but also by protecting antioxidant status, thus leading to reduced oxidative damage to lipids and proteins in AD. Therefore, by reducing oxidative stress, a walnut-enriched diet may help reduce the risk or delay the onset and progression of AD.

Methylmercury Exposure Induces Sexual Dysfunction in Male and Female Drosophila Melanogaster.

Mercury, an environmental health hazard, is a neurotoxic heavy metal. In this study, the effect of methylmercury (MeHg) exposure was analyzed on sexual behavior in Drosophila melanogaster (fruit fly), because neurons play a vital role in sexual functions. The virgin male and female flies were fed a diet mixed with different concentrations of MeHg (28.25, 56.5, 113, 226, and 339 µM) for four days, and the effect of MeHg on copulation of these flies was studied. While male and female control flies (no MeHg) and flies fed with lower concentrations of MeHg (28.25, 56.5 µM) copulated in a normal manner, male and female flies exposed to higher concentrations of MeHg (113, 226, and 339 µM) did not copulate. When male flies exposed to higher concentrations of MeHg were allowed to copulate with control female flies, only male flies fed with 113 µM MeHg were able to copulate. On the other hand, when female flies exposed to higher concentrations of MeHg were allowed to copulate with control male flies, none of the flies could copulate. After introduction of male and female flies in the copulation chamber, duration of wing flapping by male flies decreased in a MeHg-concentration-dependent manner from 101 ± 24 seconds (control) to 100.7 ± 18, 96 ±12, 59 ± 44, 31 ± 15, and 3.7 ± 2.7 seconds at 28.25, 56.5, 113, 226, and 339 µM MeHg, respectively. On the other hand, grooming in male and female flies increased in a MeHg-concentration-dependent manner. These findings suggest that MeHg exposure causes sexual dysfunction in male and female Drosophila melanogaster. Further studies showed that MeHg exposure increased oxidative stress and decreased triglyceride levels in a concentration-dependent manner in both male and female flies, suggesting that MeHg-induced oxidative stress and decreased triglyceride levels may partly contribute to sexual dysfunction in fruit flies.

Monoamine oxidase-A and B activities in the cerebellum and frontal cortex of children and young adults with autism.

Monoamine oxidases (MAOs) catalyze the metabolism of monoamine neurotransmitters, such as serotonin, dopamine, and norepinephrine, and are key regulators for brain function. In this study, we analyzed the activities of MAO-A and MAO-B in the cerebellum and frontal cortex from subjects with autism and age-matched control subjects. In the cerebellum, MAO-A activity in subjects with autism (aged 4-38 years) was significantly lower by 20.6% than in controls. When the subjects were divided into children (aged 4-12 years) and young adults (aged 13-38 years) subgroups, a significant decrease by 27.8% in the MAO-A activity was observed only in children with autism compared with controls. When the 95% confidence interval of the control group was taken as a reference range, reduced activity of MAO-A was observed in 70% of children with autism. In the frontal cortex, MAO-A activity in children with autism was also lower by 30% than in the control group, and impaired activity of MAO-A was observed in 55.6% of children with autism, although the difference between the autism and control groups was not significant when all subjects were considered. On the contrary, there was no significant difference in MAO-B activity in both the cerebellum and frontal cortex between children with autism and the control group as well as in adults. These results suggest impaired MAO-A activity in the brain of subjects with autism, especially in children with autism. Decreased activity of MAOs may lead to increased levels of monoaminergic neurotransmitters, such as serotonin, which have been suggested to have a critical role in autism. © 2017 Wiley Periodicals, Inc.

Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior.

Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1+) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1+ mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1+ mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (-/-) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1+ mice, the Atp8a1 (-/-) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.

Effects of methylmercury and alcohol exposure in Drosophila melanogaster: Potential risks in neurodevelopmental disorders.

Extensive evidence suggests the role of oxidative stress in autism and other neurodevelopmental disorders. In this study, we investigated whether methylmercury (MeHg) and/or alcohol exposure has deleterious effects in Drosophila melanogaster (fruit flies). A diet containing different concentrations of MeHg in Drosophila induced free radical generation and increased lipid peroxidation (markers of oxidative stress) in a dose-dependent manner. This effect of MeHg on oxidative stress was enhanced by further exposure to alcohol. It was observed that alcohol alone could also induce free radical generation in flies. After alcohol exposure, MeHg did not affect the immobilization of flies, but it increased the recovery time in a concentration-dependent manner. MeHg significantly inhibited the activity of alcohol dehydrogenase (ADH) in a dose-dependent manner. Linear regression analysis showed a significant negative correlation between ADH activity and recovery time upon alcohol exposure in the flies fed a diet with MeHg. This relationship between ADH activity and recovery time after alcohol exposure was confirmed by adding 4-methyl pyrazole (an inhibitor of ADH) to the diet for the flies. These results suggest that consumption of alcohol by pregnant mothers who are exposed to MeHg may lead to increased oxidative stress and to increased length of time for alcohol clearance, which may have a direct impact on the development of the fetus, thereby increasing the risk of neurodevelopmental disorders.

Effect of bisphenol A on Drosophila melanogaster behavior--a new model for the studies on neurodevelopmental disorders.

Developmental disorders such as autism and attention deficit hyperactivity disorder (ADHD) appear to have a complex etiology implicating both genetic and environmental factors. Bisphenol A (BPA), a widely used chemical in the plastic containers and in the linings of food and beverage cans, has been suggested to play a possible causative role in some developmental disorders. Here, we report behavioral modifications in Drosophila melanogaster following early exposure to BPA, which may suggest BPA as an environmental risk factor for the behavioral impairments that are the basis of diagnosis of autism and ADHD. In an open field assay with perinatally BPA-exposed and vehicle-treated control Drosophila, different parameters of locomotion (distance traveled, walking speed, spatial movement, mobility, turn angle, angular velocity and meander) were analyzed using the ethovision software. We also examined the repetitive and social interaction behaviors in these flies. In an open field assay, we identified disturbances in the locomotion patterns of BPA-exposed Drosophila that may relate to the decision-making and the motivational state of the animal. An increase in repetitive behavior was observed as an increase in the grooming behavior of Drosophila following BPA exposure. Furthermore, we also observed abnormal social interaction by the BPA-exposed flies in a social setting. These results demonstrate the effect of the environmentally prevalent risk agent BPA on the behavior of Drosophila, and suggest the practicability and the ease of using Drosophila as a model in the studies of neurobehavioral developmental disorders.

Glutathione redox imbalance in brain disorders.

PURPOSE OF REVIEW: Glutathione (GSH) is a major endogenous antioxidant. Several studies have implicated GSH redox imbalance in brain disorders. Here, we summarize current evidence on how GSH depletion and GSH-related enzyme deficit are involved in the pathology of brain disorders such as autism, schizophrenia, bipolar disorder, Alzheimer's disease, and Parkinson's disease. RECENT FINDINGS: Many studies with animal models of various brain disorders and/or with clinical samples from humans with neurodegenerative and neuropsychiatric disorders have demonstrated altered levels of GSH and oxidized glutathione (GSSG), decreased ratio of GSH/GSSG, and/or impaired expressions or activities of GSH-related enzymes in the blood or brain of these individuals. GSH depletion can lead to abnormalities in methylation metabolism and mitochondrial function. A few studies showed that a GSH deficit occurs prior to neuropathological abnormalities in these diseases. The potential therapeutic agents for brain disorders include N-acetylcysteine, liposomes encapsulated with GSH, and whey protein supplement, which can increase the GSH levels in the brain and alleviate oxidative stress-associated damage and may improve the behavior of individuals with brain diseases. SUMMARY: GSH plays an important role during the onset and progression of neuropsychiatric and neurodegenerative diseases. GSH redox imbalance may be a primary cause of these brain disorders and may be used as a biomarker for diagnosis of these diseases. N-acetylcysteine and other agents that can increase the concentration of GSH in the brain are promising approaches for the treatment of these brain disorders.

Effect of trichostatin A on gelsolin levels, proteolysis of amyloid precursor protein, and amyloid beta-protein load in the brain of transgenic mouse model of Alzheimer's disease.

In vivo and in vitro studies have shown that gelsolin is an anti-amyloidogenic protein. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the expression of gelsolin. Fibrillized amyoid beta-protein (Aß) is a key constituent of amyloid plaques in the brains of patients with Alzheimer's disease (AD). We studied the effects of TSA on the levels of gelsolin; amyloid precursor protein (APP); proteolytic enzymes (γ-secretase and ß-secretase) responsible for the production of Aß; Aß-cleaving enzymes, i.e., neprilysin (NEP) and insulin-degrading enzyme (IDE); and amyloid load in the double transgenic (Tg) APPswe/PS1(δE9) mouse model of AD. Intraperitoneal injection of TSA for two months (9-11 months of age) resulted in decreased activity of HDAC, and increased levels of gelsolin in the hippocampus and cortex of the brain in AD Tg mice as compared to vehicle-treated mice. TSA also increased the levels of γ-secretase and ß-secretase activity in the brain. However, TSA did not show any effect on the activities or the expression levels of NEP and IDE in the brain. Furthermore, TSA treatment of AD Tg mice showed no change in the amyloid load (percent of examined area occupied by amyloid plaques) in the hippocampus and cortex, suggesting that TSA treatment did not result in the reduction of amyloid load. Interestingly, TSA prevented the formation of new amyloid deposits but increased the size of existing plaques. TSA treatment did not cause any apoptosis in the brain. These results suggest that TSA increases gelsolin expression in the brain, but the pleiotropic effects of TSA negate the anti-amyloidogenic effect of gelsolin in AD Tg mice.

Bisphenol A induces oxidative stress and mitochondrial dysfunction in lymphoblasts from children with autism and unaffected siblings.

Autism is a behaviorally defined neurodevelopmental disorder. Although there is no single identifiable cause for autism, roles for genetic and environmental factors have been implicated in autism. Extensive evidence suggests increased oxidative stress and mitochondrial dysfunction in autism. In this study, we examined whether bisphenol A (BPA) is an environmental risk factor for autism by studying its effects on oxidative stress and mitochondrial function in the lymphoblasts. When lymphoblastoid cells from autistic subjects and age-matched unaffected sibling controls were exposed to BPA, there was an increase in the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential in both groups. A further subdivision of the control group into two subgroups-unaffected nontwin siblings and twin siblings-showed significantly higher ROS levels without any exposure to BPA in the unaffected twin siblings compared to the unaffected nontwin siblings. ROS levels were also significantly higher in the autism vs the unaffected nontwin siblings group. The effect of BPA on three important mtDNA genes-NADH dehydrogenase 1, NADH dehydrogenase 4, and cytochrome b-was analyzed to observe any changes in the mitochondria after BPA exposure. BPA induced a significant increase in the mtDNA copy number in the lymphoblasts from the unaffected siblings group and in the unaffected twin siblings group vs the unaffected nontwin siblings. In all three genes, the mtDNA increase was seen in 70% of the subjects. These results suggest that BPA exposure results in increased oxidative stress and mitochondrial dysfunction in the autistic subjects as well as the age-matched sibling control subjects, particularly unaffected twin siblings. Therefore, BPA may act as an environmental risk factor for autism in genetically susceptible children by inducing oxidative stress and mitochondrial dysfunction.

Dietary supplementation of walnuts improves memory deficits and learning skills in transgenic mouse model of Alzheimer's disease.

Previous in vitro studies have shown that walnut extract can inhibit amyloid-ß (Aß) fibrillization, can solubilize its fibrils, and has a protective effect against Aß-induced oxidative stress and cellular death. In this study, we analyzed the effect of dietary supplementation with walnuts on learning skills, memory, anxiety, locomotor activity, and motor coordination in the Tg2576 transgenic (tg) mouse model of Alzheimer's disease (AD-tg). From the age of 4 months, the experimental groups of AD-tg mice were fed custom-mixed diets containing 6% walnuts (T6) or 9% walnuts (T9), i.e., equivalent to 1 or 1.5 oz, respectively, of walnuts per day in humans. The control groups, i.e., AD-tg and wild-type mice, were fed a diet without walnuts (T0, Wt). These experimental and control mice were examined at the ages of 13-14 months by Morris water maze (for spatial memory and learning ability), T maze (for position discrimination learning ability), rotarod (for psychomotor coordination), and elevated plus maze (for anxiety-related behavior). AD-tg mice on the control diet (T0) showed memory deficit, anxiety-related behavior, and severe impairment in spatial learning ability, position discrimination learning ability, and motor coordination compared to the Wt mice on the same diet. The AD-tg mice receiving the diets with 6% or 9% walnuts (T6 and T9) showed a significant improvement in memory, learning ability, anxiety, and motor development compared to the AD-tg mice on the control diet (T0). There was no statistically significant difference in behavioral performance between the T6/T9 mice on walnuts-enriched diets and the Wt group on the control diet. These findings suggest that dietary supplementation with walnuts may have a beneficial effect in reducing the risk, delaying the onset, or slowing the progression of, or preventing AD.

Trichostatin A increases the levels of plasma gelsolin and amyloid beta-protein in a transgenic mouse model of Alzheimer's disease.

AIMS: Gelsolin (GSN), a multifunctional protein, binds to amyloid beta-protein (Aß), inhibits its fibrillization, solubilizes preformed Aß fibrils, and helps in its clearance from the brain. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induces the protein expression of gelsolin. In the present study, we investigated how TSA-treatment of APPswe/PS1δE9 transgenic (Tg) mice of Alzheimer's disease (AD) will affect the plasma levels of gelsolin and Aß. MAIN METHODS: TSA (5mg/kg body weight on alternate days for two months) was intraperitoneally injected to AD Tg mice. Gelsolin was measured by Western blotting and Aß was measured by enzyme-linked immunosorbent assay. KEY FINDINGS: TSA-treatment significantly increased the levels of plasma gelsolin by 1.79-fold as compared with vehicle-treated control mice (p<0.01). The levels of Aß 1-40 and Aß 1-42 in the plasma were also higher in TSA-treated mice in comparison with vehicle-treated mice. The treatment of transgenic AD mice with TSA did not affect the body weight in both male and female groups as compared to vehicle-treated animals. A positive correlation was observed between the plasma levels of gelsolin and Aß 1-40 (r=0.594, p=0.042) or Aß 1-42 (r=0.616, p=0.033) in AD Tg mice. SIGNIFICANCE: These results suggest that TSA increases the levels of plasma gelsolin and Aß in AD Tg mice, which may have implications in gelsolin-mediated clearance of Aß.

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.

Reduced activity of protein kinase C in the frontal cortex of subjects with regressive autism: relationship with developmental abnormalities.

Autism is a neurodevelopmental disorder with unknown etiology. In some cases, typically developing children regress into clinical symptoms of autism, a condition known as regressive autism. Protein kinases are essential for G-protein-coupled receptor-mediated signal transduction, and are involved in neuronal functions, gene expression, memory, and cell differentiation. Recently, we reported decreased activity of protein kinase A (PKA) in the frontal cortex of subjects with regressive autism. In the present study, we analyzed the activity of protein kinase C (PKC) in the cerebellum and different regions of cerebral cortex from subjects with regressive autism, autistic subjects without clinical history of regression, and age-matched control subjects. In the frontal cortex of subjects with regressive autism, PKC activity was significantly decreased by 57.1% as compared to age-matched control subjects (p = 0.0085), and by 65.8% as compared to non-regressed autistic subjects (p = 0.0048). PKC activity was unaffected in the temporal, parietal and occipital cortices, and in the cerebellum in both autism groups, i.e., regressive and non-regressed autism as compared to control subjects. These results suggest brain region-specific alteration of PKC activity in the frontal cortex of subjects with regressive autism. Further studies showed a negative correlation between PKC activity and restrictive, repetitive and stereotyped pattern of behavior (r = -0.084, p = 0.0363) in autistic individuals, suggesting involvement of PKC in behavioral abnormalities in autism. These findings suggest that regression in autism may be attributed, in part, to alterations in G-protein-coupled receptor-mediated signal transduction involving PKA and PKC in the frontal cortex.

Abnormal intracellular accumulation and extracellular Aß deposition in idiopathic and Dup15q11.2-q13 autism spectrum disorders.

BACKGROUND: It has been shown that amyloid ß (Aß), a product of proteolytic cleavage of the amyloid ß precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type-specific amount. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aß load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aß load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aß was mainly N-terminally truncated. Increased intraneuronal accumulation of Aß(17-40/42) in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aß accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aß(1-40/42) detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aß and an extracellular deposition of full-length Aß in nonfibrillar plaques. CONCLUSIONS/SIGNIFICANCE: The higher prevalence of excessive Aß accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aß accumulation and diffuse plaque formation.

Differences between the pattern of developmental abnormalities in autism associated with duplications 15q11.2-q13 and idiopathic autism.

The purposes of this study were to identify differences in patterns of developmental abnormalities between the brains of individuals with autism of unknown etiology and those of individuals with duplications of chromosome 15q11.2-q13 (dup[15]) and autism and to identify alterations that may contribute to seizures and sudden death in the latter. Brains of 9 subjects with dup(15), 10 with idiopathic autism, and 7 controls were examined. In the dup(15) cohort, 7 subjects (78%) had autism, 7 (78%) had seizures, and 6 (67%) had experienced sudden unexplained death. Subjects with dup(15) autism were microcephalic, with mean brain weights 300 g less (1,177 g) than those of subjects with idiopathic autism (1,477 g; p<0.001). Heterotopias in the alveus, CA4, and dentate gyrus and dysplasia in the dentate gyrus were detected in 89% of dup(15) autism cases but in only 10% of idiopathic autism cases (p < 0.001). By contrast, cerebral cortex dysplasia was detected in 50% of subjects with idiopathic autism and in no dup(15) autism cases (p<0.04). The different spectrum and higher prevalence of developmental neuropathologic findings in the dup(15) cohort than in cases with idiopathic autism may contribute to the high risk of early onset of seizures and sudden death.

Brain region-specific glutathione redox imbalance in autism.

Autism is a heterogeneous, behaviorally defined neurodevelopmental disorder. Recently, we reported a brain region-specific increase in lipid peroxidation, and deficits in mitochondrial electron transport chain complexes in autism, suggesting the role of oxidative stress and mitochondrial dysfunction in the pathophysiology of autism. However, the antioxidant status of the brain is not known in autism. Glutathione is a major endogenous antioxidant that plays a crucial role in protecting cells from exogenous and endogenous toxins, particularly in the central nervous system. The present study examines the concentrations of glutathione (GSH, reduced form; and GSSG, oxidized form) and the redox ratio of GSH to GSSG (marker of oxidative stress) in different regions of brains from autistic subjects and age-matched control subjects. In the cerebellum and temporal cortex from subjects with autism, GSH levels were significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG by 38.2 and 45.5 %, respectively, as compared to the control group. There was also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism. In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism versus control group. The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism. These findings indicate that autism is associated with deficits in glutathione antioxidant defense in selective regions of the brain. We suggest that disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.