Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes

Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status

Treatments targeting metabolic abnormalities in children with autism are limited. Previously we reported that a nutritional treatment significantly improved glutathione metabolism in children with autistic disorder. In this study we evaluated changes in adaptive behaviors in this cohort and determined whether such changes are related to changes in glutathione metabolism. Thirty-seven children diagnosed with autistic disorder and abnormal glutathione and methylation metabolism were treated with twice weekly 75 µg/Kg methylcobalamin and twice daily 400 µg folinic acid for 3 months in an open-label fashion. The Vineland Adaptive Behavior Scale (VABS) and glutathione redox metabolites were measured at baseline and at the end of the treatment period. Over the treatment period, all VABS subscales significantly improved with an average effect size of 0.59, and an average improvement in skills of 7.7 months. A greater improvement in glutathione redox status was associated with a greater improvement in expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills. Age, gender, and history of regression did not influence treatment response. The significant behavioral improvements observed and the relationship between these improvements to glutathione redox status suggest that nutritional interventions targeting redox metabolism may benefit some children with autism

The aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα-and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα-and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.Scientific and Technological Research Council of Turkey (TUBITAK 106T417

Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort.

There is increasing recognition that mitochondrial dysfunction is associated with the autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction or how mitochondrial abnormalities might interact with other physiological disturbances associated with autism, such as oxidative stress. In the current study we used respirometry to examine reserve capacity, a measure of the mitochondrial ability to respond to physiological stress, in lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) as well as age and gender-matched control LCLs. We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS). Specifically, the AD LCLs exhibit a higher reserve capacity at baseline and a sharper depletion of reserve capacity when ROS exposure is increased, as compared to control LCLs. Detailed investigation indicated that reserve capacity abnormalities seen in AD LCLs were the result of higher ATP-linked respiration and maximal respiratory capacity at baseline combined with a marked increase in proton leak respiration as ROS was increased. We further demonstrate that these reserve capacity abnormalities are driven by a subgroup of eight (32%) of 25 AD LCLs. Additional investigation of this subgroup of AD LCLs with reserve capacity abnormalities revealed that it demonstrated a greater reliance on glycolysis and on uncoupling protein 2 to regulate oxidative stress at the inner mitochondria membrane. This study suggests that a significant subgroup of AD children may have alterations in mitochondrial function which could render them more vulnerable to a pro-oxidant microenvironment derived from intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxicants. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors

Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism

The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism

Dietary Intake and Plasma Levels of Choline and Betaine in Children with Autism Spectrum Disorders

Abnormalities in folate-dependent one-carbon metabolism have been reported in many children with autism. Because inadequate choline and betaine can negatively affect folate metabolism and in turn downstream methylation and antioxidant capacity, we sought to determine whether dietary intake of choline and betaine in children with autism was adequate to meet nutritional needs based on national recommendations. Three-day food records were analyzed for 288 children with autism (ASDs) who participated in the national Autism Intervention Research Network for Physical Health (AIR-P) Study on Diet and Nutrition in children with autism. Plasma concentrations of choline and betaine were measured in a subgroup of 35 children with ASDs and 32 age-matched control children. The results indicated that 60–93% of children with ASDs were consuming less than the recommended Adequate Intake (AI) for choline. Strong positive correlations were found between dietary intake and plasma concentrations of choline and betaine in autistic children as well as lower plasma concentrations compared to the control group. We conclude that choline and betaine intake is inadequate in a significant subgroup of children with ASDs and is reflected in lower plasma levels. Inadequate intake of choline and betaine may contribute to the metabolic abnormalities observed in many children with autism and warrants attention in nutritional counseling

Maternal metabolic profile predicts high or low risk of an autism pregnancy outcome

BackgroundCurrently there is no test for pregnant mothers that can predict the probability of having a child that will be diagnosed with autism spectrum disorder (ASD). Recent estimates indicate that if a mother has previously had a child with ASD, the risk of having a second child with ASD is ~18.7% (High Risk) whereas the risk of ASD in the general population is ~1.7% (Low Risk).MethodsIn this study, metabolites of the folate-dependent transmethylation and transsulfuration biochemical pathways of pregnant mothers were measured to determine whether or not the risk of having a child with autism could be predicted by her metabolic profile. Pregnant mothers who have had a child with autism before were separated into two groups based on the diagnosis of their child whether the child had autism (ASD) or not (TD). Then these mothers were compared to a group of control mothers who have not had a child with autism before. A total of 107 mothers were in the High Risk category and 25 mothers in the Low Risk category. The High Risk category was further separated into 29 mothers in the ASD group and 78 mothers in the TD group.ResultsThe metabolic results indicated that among High Risk mothers, it was not possible to predict an autism pregnancy outcome. However, the metabolic profile was able to predict with approximately 90% sensitivity and specificity whether a mother fell into the High Risk group (18.7% risk) or Low Risk group (1.7% risk).ConclusionsBased upon these measurements it is not possible to determine during a pregnancy if a child will be diagnosed with ASD by age 3. However, differences in the folate-dependent transmethylation and transsulfuration metabolites are indicative of the risk level (High Risk of 18.7% vs. Low Risk of 1.7%) of the mother for having a child with ASD

The Seahorse assay.

<p>Oxygen consumption rate (OCR) is measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. Initially, baseline cellular OCR is measured, from which basal respiration can be derived by subtracting non-mitochondrial respiration. Next oligomycin, a complex V inhibitor, is added and the resulting OCR is used to derive ATP-linked respiration (by subtracting the oligomycin rate from baseline cellular OCR) and proton leak respiration (by subtracting non-mitochondrial respiration from the oligomycin rate). Next carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP), a protonophore, is added to collapse the inner membrane gradient, allowing the ETC to function at its maximal rate, and maximal respiratory capacity is derived by subtracting non-mitochondrial respiration from the FCCP rate. Lastly, antimycin A and rotenone, inhibitors of complex III and I, are added to shut down ETC function, revealing the non-mitochondrial respiration. Mitochondrial reserve capacity is calculated by subtracting basal respiration from maximal respiratory capacity.</p

Mitochondrial respiratory parameters and responses to DMNQ differ in two AD LCL subgroups.

<p>Overall, the AD-N subgroup (A–D) demonstrates similar mitochondrial responses as the control LCLs while the AD-A subgroup (E–H) parallels the differences between the AD and control LCLs found in the overall analysis. For the AD-N subgroup (<b>A</b>) ATP-linked respiration and (<b>D</b>) reserve capacity were overall slightly but significantly lower in the AD-N LCLs while (<b>B</b>) proton leak respiration was overall slightly but significantly higher in the AD-N LCLs, and (<b>C</b>) maximal respiratory capacity was not different in the AD-N LCLs as compared to controls. For the AD-A subgroup, (<b>E</b>) ATP-linked respiration, (<b>F</b>) proton leak respiration and (<b>G</b>) maximal respiratory capacity were overall markedly higher for AD-A LCLs as compared to control LCLs. (<b>H</b>) Reserve capacity was significantly greater for the AD-A LCLs as compared to control LCLs at baseline but decreased such that it was significantly lower than controls at 10–15 µM DMNQ. (<b>I</b>) ATP-linked respiration was overall markedly higher for AD-A LCLs as compared to AD-N LCLs. (<b>J</b>) Proton leak respiration was significantly higher in the AD-A LCLs as compared to the AD-N LCLs at 5–15 µM DMNQ. (<b>K</b>) Maximal respiratory capacity was significantly higher for AD-A LCLs as compared to AD-N LCLs at baseline and 5 µM DMNQ. (<b>L</b>) Reserve capacity was significantly greater for the AD-A LCLs at baseline but decreased so that it was significantly lower for the AD-A LCLs as compared to the AD-N LCLs at 12.5 and 15 µM DMNQ. *p<0.001; **p<0.0001; # p<0.01; p<0.05; ↕ indicates an overall statistical difference between LCL groups.</p

DMNQ exposure alters the glutathione redox status of LCLs.

<p>The change in intracellular (<b>A</b>) reduced glutathione (GSH), (<b>B</b>) oxidized glutathione (GSSG) and (<b>C</b>) the reduced-to-oxidized glutathione ratio (GSH/GSSG) with increased intracellular oxidative stress was measured in control (n = 3) and AD (n = 5) LCLs treated with indicated concentrations of the redox cycling agent DMNQ for 1 h. Results are expressed per mg protein. Overall DMNQ significantly reduces GSH and GSH/GSSH and increases GSSG.</p