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.

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.

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.

Consensus paper: pathological role of the cerebellum in autism.

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene-environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

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.

Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism.

Mitochondria play important roles in generation of free radicals, ATP formation, and in apoptosis. We studied the levels of mitochondrial electron transport chain (ETC) complexes, that is, complexes I, II, III, IV, and V, in brain tissue samples from the cerebellum and the frontal, parietal, occipital, and temporal cortices of subjects with autism and age-matched control subjects. The subjects were divided into two groups according to their ages: Group A (children, ages 4-10 years) and Group B (adults, ages 14-39 years). In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p<0.05), of complex I in the frontal cortex (p<0.05), and of complexes II (p<0.01), III (p<0.01), and V (p<0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism. In the cerebellum and temporal cortex, no overlap was observed in the levels of these ETC complexes between subjects with autism and control subjects. In the frontal cortex of Group A, a lower level of ETC complexes was observed in a subset of autism cases, that is, 60% (3/5) for complexes I, II, and V, and 40% (2/5) for complexes III and IV. A striking observation was that the levels of ETC complexes were similar in adult subjects with autism and control subjects (Group B). A significant increase in the levels of lipid hydroperoxides, an oxidative stress marker, was also observed in the cerebellum and temporal cortex in the children with autism. These results suggest that the expression of ETC complexes is decreased in the cerebellum and the frontal and temporal regions of the brain in children with autism, which may lead to abnormal energy metabolism and oxidative stress. The deficits observed in the levels of ETC complexes in children with autism may readjust to normal levels by adulthood.

Protective effects of walnut extract against amyloid beta peptide-induced cell death and oxidative stress in PC12 cells.

Amyloid beta-protein (Aß) is the major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer's disease. Aß is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Recently, considerable attention has been focused on dietary antioxidants that are able to scavenge reactive oxygen species (ROS), thereby offering protection against oxidative stress. Walnuts are rich in components that have anti-oxidant and anti-inflammatory properties. The inhibition of in vitro fibrillization of synthetic Aß, and solubilization of preformed fibrillar Aß by walnut extract was previously reported. The present study was designed to investigate whether walnut extract can protect against Aß-induced oxidative damage and cytotoxicity. The effect of walnut extract on Aß-induced cellular damage, ROS generation and apoptosis in PC12 pheochromocytoma cells was studied. Walnut extract reduced Aß-mediated cell death assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, and release of lactate dehydrogenase (membrane damage), DNA damage (apoptosis) and generation of ROS in a concentration-dependent manner. These results suggest that walnut extract can counteract Aß-induced oxidative stress and associated cell death.

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.

BDNF-Akt-Bcl2 antiapoptotic signaling pathway is compromised in the brain of autistic subjects.

Although the pathogenesis of autism is not understood, emerging evidence points to apoptotic mechanisms being involved in this disorder. However, it is not known whether apoptosis signaling is deregulated in the brain of autistic subjects. This study investigates how the apoptosis-related proteins are regulated in the autistic brain. Our studies show that Bcl2 is significantly decreased, whereas the expression of p53 is increased, in the brain of autistic subjects in comparison with age-matched controls. We also found that the expression and phosphorylation/activation of Akt kinase that regulates Bcl2 are significantly decreased in the autistic brain. The down-regulation of Akt may result from a decreased concentration of brain-derived neurotrophic factor (BDNF), the growth factor that modulates Akt activities. These results suggest that down-regulation of the BDNF-Akt-Bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism.

The neuropathology of autism: defects of neurogenesis and neuronal migration, and dysplastic changes.

Autism is characterized by a broad spectrum of clinical manifestations including qualitative impairments in social interactions and communication, and repetitive and stereotyped patterns of behavior. Abnormal acceleration of brain growth in early childhood, signs of slower growth of neurons, and minicolumn developmental abnormalities suggest multiregional alterations. The aim of this study was to detect the patterns of focal qualitative developmental defects and to identify brain regions that are prone to developmental alterations in autism. Formalin-fixed brain hemispheres of 13 autistic (4-60 years of age) and 14 age-matched control subjects were embedded in celloidin and cut into 200-mum-thick coronal sections, which were stained with cresyl violet and used for neuropathological evaluation. Thickening of the subependymal cell layer in two brains and subependymal nodular dysplasia in one brain is indicative of active neurogenesis in two autistic children. Subcortical, periventricular, hippocampal and cerebellar heterotopias detected in the brains of four autistic subjects (31%) reflect abnormal neuronal migration. Multifocal cerebral dysplasia resulted in local distortion of the cytoarchitecture of the neocortex in four brains (31%), of the entorhinal cortex in two brains (15%), of the cornu Ammonis in four brains and of the dentate gyrus in two brains. Cerebellar flocculonodular dysplasia detected in six subjects (46%), focal dysplasia in the vermis in one case, and hypoplasia in one subject indicate local failure of cerebellar development in 62% of autistic subjects. Detection of flocculonodular dysplasia in only one control subject and of a broad spectrum of focal qualitative neuropathological developmental changes in 12 of 13 examined brains of autistic subjects (92%) reflects multiregional dysregulation of neurogenesis, neuronal migration and maturation in autism, which may contribute to the heterogeneity of the clinical phenotype.

Calcium induces expression of cytoplasmic gelsolin in SH-SY5Y and HEK-293 cells.

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.

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.

Elevated immune response in the brain of autistic patients.

UNLABELLED: This study determined immune activities in the brain of ASD patients and matched normal subjects by examining cytokines in the brain tissue. Our results showed that proinflammatory cytokines (TNF-alpha, IL-6 and GM-CSF), Th1 cytokine (IFN-gamma) and chemokine (IL-8) were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients. CONCLUSION: ASD patients displayed an increased innate and adaptive immune response through the Th1 pathway, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD.

Gelsolin levels are increased in the brain as a function of age during normal development in children that are further increased in Down syndrome.

Neuronal dysfunctions in several neurodegenerative diseases such as Down syndrome (DS) have been linked to oxidative stress. In this study, we observed that lipid peroxidation, a marker of oxidative stress, is significantly increased in the frontal cortex of brains of individuals with DS as compared with control subjects. We report here that gelsolin levels are increased in the frontal cortex of individuals with DS as compared with controls during early developmental ages (5 to 13 y). Interestingly, the levels of gelsolin in the frontal cortex were increased as a function of age in both DS and control subjects. Because cytoplasmic gelsolin has 5 free thiol groups (cysteine), and its levels are increased in response to oxidative stress, we propose that gelsolin may serve as an antioxidant protein.

Cytoplasmic gelsolin in pheochromocytoma-12 cells forms a complex with amyloid beta-protein.

Gelsolin exists as secretory (plasma) and cytoplasmic forms. We have reported that plasma gelsolin binds to amyloid beta-protein (Abeta), and inhibits its fibrillization. Others reported that peripheral administration or transgene expression of plasma gelsolin reduces amyloid load in transgenic mouse models of Alzheimer's disease. Here, we report that the expression of cytoplasmic gelsolin in pheochromocytoma-12 cells increases after treatment with hydrogen peroxide. When synthetic Abeta was fortified with cell lysate, cytoplasmic gelsolin co-immunoprecipitated with Abeta. The results suggest that cytoplasmic gelsolin forms a complex with Abeta in a manner like plasma form, and it may also regulate Abeta fibrillization. This report indicates that structural differences between plasma and cytoplasmic gelsolin do not play a key role in their complex formation with Abeta.

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.

Binding of proteases to fibrillar amyloid-beta protein and its inhibition by Congo red.

Fibrillar amyloid-beta protein (fAbeta) is the principal component of amyloid plaques in the brains of patients with Alzheimer's disease (AD). We have recently reported that activity of trypsin is inhibited by fAbeta and that trypsin can bind to fAbeta. Neprilysin and insulysin are important proteases for the clearance of soluble Abeta. Here, we report that fAbeta also binds to neprilysin and insulysin, which results in the inhibition of their proteolytic activities. These findings suggest that clearance of soluble Abeta may be defective in AD because of binding of proteases to amyloid plaques, leading to inactivation of proteases that are required for catabolism of Abeta. The identification of compounds that can inhibit binding of proteases to fAbeta may, therefore, be of significance for therapeutic intervention in AD. Congo red and Thioflavin T are widely used for histopathological examination of amyloid plaques because of their strong affinity to fibrillar amyloid proteins. We examined the effect of Congo red and Thioflavin T (potent fAbeta-binding compounds) on the binding of different proteases to fAbeta. While Congo red inhibited the binding of trypsin, neprilysin and insulysin to fAbeta, Thioflavin T did not have any effect. The effect of Congo red was concentration-dependent and the inhibitory effect was in the order of trypsin > insulysin > neprilysin. When the effect of prebound-Congo red to fAbeta was examined, trypsin was unable to bind to this complex suggesting that Congo red may have better affinity than trypsin for binding to fAbeta. Based on these results, we propose that the inhibition of binding of proteases to amyloid plaques may help in reducing the deposition of Abeta in 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.

Binding of trypsin to fibrillar amyloid beta-protein.

We have recently reported that fibrillar amyloid beta-protein (Abeta) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Abeta (fAbeta) and the resulting complex of trypsin/fAbeta is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Abeta 1-40 and Abeta 1-42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAbeta sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAbeta that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Abeta was dependent on the degree of Abeta fibrillization and on the concentration of fAbeta. However, the trypsin binding to Abeta oligomers did not affect the fibril growth. The maximum binding (B(max)) of trypsin to fAbeta 1-40 and fAbeta 1-42 was 36 pmol and 40 pmol, and dissociation constant (K(d)) was 18.31 microM and 20 microM respectively. Similar to fAbeta, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Abeta. These results suggest that fAbeta and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.

Oxidative stress in autism.

Autism is a severe developmental disorder with poorly understood etiology. Oxidative stress in autism has been studied at the membrane level and also by measuring products of lipid peroxidation, detoxifying agents (such as glutathione), and antioxidants involved in the defense system against reactive oxygen species (ROS). Lipid peroxidation markers are elevated in autism, indicating that oxidative stress is increased in this disease. Levels of major antioxidant serum proteins, namely transferrin (iron-binding protein) and ceruloplasmin (copper-binding protein), are decreased in children with autism. There is a positive correlation between reduced levels of these proteins and loss of previously acquired language skills in children with autism. The alterations in ceruloplasmin and transferrin levels may lead to abnormal iron and copper metabolism in autism. The membrane phospholipids, the prime target of ROS, are also altered in autism. The levels of phosphatidylethanolamine (PE) are decreased, and phosphatidylserine (PS) levels are increased in the erythrocyte membrane of children with autism as compared to their unaffected siblings. Several studies have suggested alterations in the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase in autism. Additionally, altered glutathione levels and homocysteine/methionine metabolism, increased inflammation, excitotoxicity, as well as mitochondrial and immune dysfunction have been suggested in autism. Furthermore, environmental and genetic factors may increase vulnerability to oxidative stress in autism. Taken together, these studies suggest increased oxidative stress in autism that may contribute to the development of this disease. A mechanism linking oxidative stress with membrane lipid abnormalities, inflammation, aberrant immune response, impaired energy metabolism and excitotoxicity, leading to clinical symptoms and pathogenesis of autism is proposed.