Overexpression of LINE-1 Retrotransposons in Autism Brain.

Long interspersed nuclear elements-1 (LINE-1 or L1) are mobile DNA sequences that are capable of duplication and insertion (retrotransposition) within the genome. Recently, retrotransposition of L1 was shown to occur within human brain leading to somatic mosaicism in hippocampus and cerebellum. Because unregulated L1 activity can promote genomic instability and mutagenesis, multiple mechanisms including epigenetic chromatin condensation have evolved to effectively repress L1 expression. Nonetheless, L1 expression has been shown to be increased in patients with Rett syndrome and schizophrenia. Based on this evidence and our reports of oxidative stress and epigenetic dysregulation in autism cerebellum, we sought to determine whether L1 expression was increased in autism brain. The results indicated that L1 expression was significantly elevated in the autism cerebellum but not in BA9, BA22, or BA24. The binding of repressive MeCP2 and histone H3K9me3 to L1 sequences was significantly lower in autism cerebellum suggesting that relaxation of epigenetic repression may have contributed to increased expression. Further, the increase in L1 expression was inversely correlated with glutathione redox status consistent with reports indicating that L1 expression is increased under pro-oxidant conditions. Finally, the expression of transcription factor FOXO3, sensor of oxidative stress, was significantly increased and positively associated with L1 expression and negatively associated with glutathione redox status. While these novel results are an important first step, future understanding of the contribution of elevated L1 expression to neuronal CNVs and genomic instability in autism will depend on emerging cell-specific genomic technologies, a challenge that warrants future investigation.

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

BACKGROUND: Currently 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). METHODS: In 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. RESULTS: The 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). CONCLUSIONS: Based 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.

Short-term dietary methionine supplementation affects one-carbon metabolism and DNA methylation in the mouse gut and leads to altered microbiome profiles, barrier function, gene expression and histomorphology.

BACKGROUND: Methionine, a central molecule in one-carbon metabolism, is an essential amino acid required for normal growth and development. Despite its importance to biological systems, methionine is toxic when administered at supra-physiological levels. The aim of this study was to investigate the effects of short-term methionine dietary modulation on the proximal jejunum, the section of the gut specifically responsible for amino acid absorption, in a mouse model. Eight-week-old CBA/J male mice were fed methionine-adequate (MAD; 6.5 g/kg) or methionine-supplemented (MSD; 19.5 g/kg) diets for 3.5 or 6 days (average food intake 100 g/kg body weight). The study design was developed in order to address the short-term effects of the methionine supplementation that corresponds to methionine dietary intake in Western populations. Biochemical indices in the blood as well as metabolic, epigenetic, transcriptomic, metagenomic, and histomorphological parameters in the gut were evaluated. RESULTS: By day 6, feeding mice with MSD (protein intake <10% different from MAD) resulted in increased plasma (2.3-fold; p < 0.054), but decreased proximal jejunum methionine concentrations (2.2-fold; p < 0.05) independently of the expression of neutral amino acid transporters. MSD has also caused small bowel bacteria colonization, increased the abundance of pathogenic bacterial species Burkholderiales and decreased the gene expression of the intestinal transmembrane proteins-Cldn8 (0.18-fold, p < 0.05), Cldn9 (0.24-fold, p < 0.01) and Cldn10 (0.05-fold, p < 0.05). Feeding MSD led to substantial histomorphological alterations in the proximal jejunum exhibited as a trend towards decreased plasma citrulline concentrations (1.8-fold, p < 0.07), as well as loss of crypt depth (by 28%, p < 0.05) and mucosal surface (by 20%, p < 0.001). CONCLUSIONS: Together, these changes indicate that short-term feeding of MSD substantially alters the normal gut physiology. These effects may contribute to the pathogenesis of intestinal inflammatory diseases and/or sensitize the gut to exposure to other stressors.

Effects of Obesity on Pro-Oxidative Conditions and DNA Damage in Liver of DMBA-Induced Mammary Carcinogenesis Models.

The prevalence of the overweight and obesity is on the rise worldwide. Obesity can increase the risk of certain cancers and liver steatosis development. Previously, we reported that obesity increased liver steatosis in a mammary tumor model, but little is known about the effects of obesity in the liver in regard to global DNA methylation, DNA damage, and oxidative/nitrosative stress. Using a mammary tumor model, we investigated the effects of obesity on oxidative stress and DNA reaction. Five-week-old lean and obese female rats were used. At 50 days of age, all rats received 7,12-dimethylbenz(α)anthracene (DMBA) and were sacrificed 155 days later. HPLC with electrochemical and ultraviolet detection and LC-MS were used. Obesity caused higher (p < 0.0004) methionine levels, had no effect (p < 0.055) on SAM levels, caused lower (p < 0.0005) SAH levels, caused higher (p < 0.0005) SAM/SAH ratios, and increased (p < 0.02) global DNA methylation. Levels of free reduced GSH were not significantly lower (p < 0.08), but free oxidized GSSG was higher (p < 0.002) in obese rats. The GSH/GSSG ratio was lower (p < 0.0001), and oxidized guanosine was higher (p < 0.002) in DNA of obese rats compared to lean rats. Obesity caused significant oxidative/nitrosative stress, oxidative DNA damage, and change of DNA methylation pattern in the liver, and these changes may contribute to the development of liver steatosis in breast cancer models.

Effects of local irradiation combined with sunitinib on early remodeling, mitochondria, and oxidative stress in the rat heart.

BACKGROUND AND PURPOSE: Thoracic (chemo)radiation therapy is increasingly administered with tyrosine kinase inhibitors (TKI). While TKI have adverse effects on the heart, it is unknown whether combination with other cancer therapies causes enhanced toxicity. We used an animal model to investigate whether radiation and sunitinib interact in their effects on the heart. MATERIAL AND METHODS: Male Sprague-Dawley rats received local heart irradiation (9Gy per day, 5days). Oral sunitinib (8 or 15mg/kg bodyweight per day) started on day 1 of irradiation and continued for 2weeks. Cardiac function was examined with echocardiography. Cardiac remodeling, cell death, left ventricular (LV) oxidative stress markers, mitochondrial morphology and mitochondrial permeability transition pore (mPTP) opening were assessed. RESULTS: Cardiac diameter, stroke volume, and LV volume, mass and anterior wall thickness increased in time, but only in the vehicle group. Sunitinib reduced LV inner diameter and volume in systole, which were counteracted by radiation. Sunitinib and radiation showed enhanced effects on mitochondrial morphology and mPTP opening, but not on cardiac troponin I, mast cell numbers or markers of oxidative stress. CONCLUSIONS: This study found no early enhanced effects of radiation and sunitinib on cardiac function or structure. Long-term effects remain to be determined.

Radiation-induced changes in DNA methylation of repetitive elements in the mouse heart.

DNA methylation is a key epigenetic mechanism, needed for proper control over the expression of genetic information and silencing of repetitive elements. Exposure to ionizing radiation, aside from its strong genotoxic potential, may also affect the methylation of DNA, within the repetitive elements, in particular. In this study, we exposed C57BL/6J male mice to low absorbed mean doses of two types of space radiation-proton (0.1 Gy, 150 MeV, dose rate 0.53 ± 0.08 Gy/min), and heavy iron ions ((56)Fe) (0.5 Gy, 600 MeV/n, dose rate 0.38 ± 0.06 Gy/min). Radiation-induced changes in cardiac DNA methylation associated with repetitive elements were detected. Specifically, modest hypomethylation of retrotransposon LINE-1 was observed at day 7 after irradiation with either protons or (56)Fe. This was followed by LINE-1, and other retrotransposons, ERV2 and SINE B1, as well as major satellite DNA hypermethylation at day 90 after irradiation with (56)Fe. These changes in DNA methylation were accompanied by alterations in the expression of DNA methylation machinery and affected the one-carbon metabolism pathway. Furthermore, loss of transposable elements expression was detected in the cardiac tissue at the 90-day time-point, paralleled by substantial accumulation of mRNA transcripts, associated with major satellites. Given that the one-carbon metabolism pathway can be modulated by dietary modifications, these findings suggest a potential strategy for the mitigation and, possibly, prevention of the negative effects exerted by ionizing radiation on the cardiovascular system. Additionally, we show that the methylation status and expression of repetitive elements may serve as early biomarkers of exposure to space radiation.

A tocotrienol-enriched formulation protects against radiation-induced changes in cardiac mitochondria without modifying late cardiac function or structure.

Radiation-induced heart disease (RIHD) is a common and sometimes severe late side effect of radiation therapy for intrathoracic and chest wall tumors. We have previously shown that local heart irradiation in a rat model caused prolonged changes in mitochondrial respiration and increased susceptibility to mitochondrial permeability transition pore (mPTP) opening. Because tocotrienols are known to protect against oxidative stress-induced mitochondrial dysfunction, in this study, we examined the effects of tocotrienols on radiation-induced alterations in mitochondria, and structural and functional manifestations of RIHD. Male Sprague-Dawley rats received image-guided localized X irradiation to the heart to a total dose of 21 Gy. Twenty-four hours before irradiation, rats received a tocotrienol-enriched formulation or vehicle by oral gavage. Mitochondrial function and mitochondrial membrane parameters were studied at 2 weeks and 28 weeks after irradiation. In addition, cardiac function and histology were examined at 28 weeks. A single oral dose of the tocotrienol-enriched formulation preserved Bax/Bcl2 ratios and prevented mPTP opening and radiation-induced alterations in succinate-driven mitochondrial respiration. Nevertheless, the late effects of local heart irradiation pertaining to myocardial function and structure were not modified. Our studies suggest that a single dose of tocotrienols protects against radiation-induced mitochondrial changes, but these effects are not sufficient against long-term alterations in cardiac function or remodeling.

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.

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.

Complex epigenetic regulation of engrailed-2 (EN-2) homeobox gene in the autism cerebellum.

The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.

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.

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.

Metabolic imbalance associated with methylation dysregulation and oxidative damage in children with autism.

Oxidative stress and abnormal DNA methylation have been implicated in the pathophysiology of autism. We investigated the dynamics of an integrated metabolic pathway essential for cellular antioxidant and methylation capacity in 68 children with autism, 54 age-matched control children and 40 unaffected siblings. The metabolic profile of unaffected siblings differed significantly from case siblings but not from controls. Oxidative protein/DNA damage and DNA hypomethylation (epigenetic alteration) were found in autistic children but not paired siblings or controls. These data indicate that the deficit in antioxidant and methylation capacity is specific for autism and may promote cellular damage and altered epigenetic gene expression. Further, these results suggest a plausible mechanism by which pro-oxidant environmental stressors may modulate genetic predisposition to autism.

A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism.

The biologic basis of autism is complex and is thought to involve multiple and variable gene-environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate-dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one-carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell-specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population-based sample of autism case-parent triads. In case-control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S-adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism.

Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism.

Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.

Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism.

BACKGROUND: Metabolic abnormalities and targeted treatment trials have been reported for several neurobehavioral disorders but are relatively understudied in autism. OBJECTIVE: The objective of this study was to determine whether or not treatment with the metabolic precursors, methylcobalamin and folinic acid, would improve plasma concentrations of transmethylation/transsulfuration metabolites and glutathione redox status in autistic children. DESIGN: In an open-label trial, 40 autistic children were treated with 75 microg/kg methylcobalamin (2 times/wk) and 400 microg folinic acid (2 times/d) for 3 mo. Metabolites in the transmethylation/transsulfuration pathway were measured before and after treatment and compared with values measured in age-matched control children. RESULTS: The results indicated that pretreatment metabolite concentrations in autistic children were significantly different from values in the control children. The 3-mo intervention resulted in significant increases in cysteine, cysteinylglycine, and glutathione concentrations (P < 0.001). The oxidized disulfide form of glutathione was decreased and the glutathione redox ratio increased after treatment (P < 0.008). Although mean metabolite concentrations were improved significantly after intervention, they remained below those in unaffected control children. CONCLUSION: The significant improvements observed in transmethylation metabolites and glutathione redox status after treatment suggest that targeted nutritional intervention with methylcobalamin and folinic acid may be of clinical benefit in some children who have autism. This trial was registered at (clinicaltrials.gov) as NCT00692315.

Induction of oxidative stress and DNA damage in rat brain by a folate/methyl-deficient diet.

The age-associated decline in cellular antioxidant defenses and resultant accumulation of DNA damage in central nervous system has been mechanistically implicated in the etiology and pathogenesis of neurodegenerative diseases. Neurons possess a high metabolic activity and are especially vulnerable to the long-term effects of continuous exposure to endogenous reactive oxygen species. It is well recognized that adequate availability of essential nutrients involved in cellular one-carbon metabolism is essential for normal brain development and function. Additionally, the synthesis of the primary low-molecular cellular antioxidant glutathione is inter-dependently linked to one-carbon metabolic pathway. Thus, any aberrant disruptions in one-carbon metabolism can result in potentially deleterious effects including cell death as a result of an imbalance in the cellular redox state. Hence, in the present study, we examined the long-term effects of a folate/methyl-deficient (FMD) diet on cellular antioxidant defenses and DNA damage in the rat brain. Feeding male Fisher 344 rats a FMD diet resulted in perturbations in the levels of one-carbon metabolites along with induction of oxidative stress and oxidative DNA damage in the brain. This was evidenced by a decrease in the reduced oxidized/glutathione ratio, imbalance of cellular antioxidant defense system; specifically, altered activity and expression of antioxidant enzymes Mn-containing superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPX), increased accumulation of oxidative DNA lesions, 8-hydroxydeoxyguanosine (8-OH-dG) and DNA single-strand breaks, even in the presence of increased expression of critical DNA repair genes apurinic/apyrimidinic endonuclease 1 (Apex1) and DNA polymerase beta (Polbeta), and apoptosis in the brains of folate/methyl-deficient rats. These results indicate that chronic methyl group deficiency leads to an imbalance in cellular antioxidant defense systems, increased oxidative stress, and apoptosis. Any of these events may compromise normal central nervous system function and contribute to the development of various neurological, behavioral, and neurocognitive dysfunctions.

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.

Cog1p plays a central role in the organization of the yeast conserved oligomeric Golgi complex.

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved peripheral membrane oligomeric protein complex that is involved in intra-Golgi protein trafficking. The COG complex is composed of eight subunits that are located in two lobes; Lobe A contains COG1-4, and Lobe B is composed of COG5-8. Both in vivo and in vitro protein-protein interaction techniques were applied to characterize interactions between individual COG subunits. In vitro assays revealed binary interactions between Cog2p and Cog3p, Cog2p and Cog4p, and Cog6p and Cog8p and a strong interaction between Cog5p and Cog7p. The two-hybrid assay confirmed these findings and revealed that Cog1p interacted with subunits from both lobes of the complex. Antibodies to COG subunits were utilized to determine the protein levels and membrane association of COG subunits in yeast delta cog1-8 mutants. As a result, we created a model of the protein-protein interactions within the yeast COG complex and proposed that Cog1p is a bridging subunit between the two COG lobes. In support of this hypothesis, we have demonstrated that Cog1p is required for the stable association between two COG subcomplexes.