Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies. Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice. More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.http://dx.doi.org/10.1371/journal.pmed.0060001.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Rene Toes and Tom HuizingaThis study is further discussed in a rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)The MedlinePlus Encyclopedia has a page on rheumatoid arthritisThe UK National Health Service Choices information service has detailed information on Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritisThe US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides rheumatoid arthritis that includes statistics about this disease and its impact on daily lifeThe US Centers for Disease Control and Prevention has an overview on  Rheumatoid arthritis (RA) is a chronic inflammatory erosive polyarthritis with an associated significant morbidity and mortality . One of Physiologically, antigen-driven antibody responses take place within germinal centres (GCs) of secondary lymphoid organs where the processes of somatic hypermutation (SHM) and class switch recombination (CSR) of the Ig genes in GC-B cells occur, leading to affinity maturation and differentiation to memory B and plasma cells ,14. HoweThe presence of ectopic lymphoid structures resembling GCs and characterized by follicular dendritic cell (FDC) networks is a common finding in chronically inflamed tissues in several autoimmune diseases  includinThus, the purpose of this study was to investigate whether ectopic lymphoid structures in RA synovium are functional by determining whether they: (i) express AID, the enzyme required for SHM and CSR of Ig genes; (ii) support ongoing CSR and the production of high-affinity ACPA; and (iii) remain functional and promote B cell survival, proliferation, and autoantibody production in an RA/SCID chimera model devoid of any new immune cell influx into the synovium and where the ongoing immunological activity of secondary lymphoid organs is excluded.Following written informed consent, synovial tissue was collected from 55 patients with RA fulfilling the revised 1987 ACR criteria for RA . TissuesFrom 24 of the 55 patients who underwent joint replacement surgery between 1999 and 2005, only formaldehyde fixed, paraffin-embedded tissue was available and therefore was exclusively used for histological analysis. From an additional 25 patients (of whom ten underwent arthroplastic surgery and 15 underwent ultrasound-guided biopsy), enrolled between 2005 and 2008, paired paraffin and RNA samples were available and were used for histological analysis and QT-PCR. Finally, from six patients with RA undergoing joint replacement surgery, synovial tissue was obtained and stored in liquid nitrogen until use for transplantation into SCID mice.Procedures were approved by the hospital Ethics Committee (REC/98/11/27 Guys and St. Thomas' NHS Trust and REC 05/Q0703/198 Barts and the London NHS Trust) and performed after written informed consent.A list of primary and secondary antibodies (Abs) used is reported in The histological grading of tissues and degree of lymphoid organization was assessed by immunohistochemical staining of sequentially cut sections of RA synovial tissue, as previously reported . BrieflyStaining for CD3 and CD20 (using antibody dilutions of 1:50 and 1:20 respectively), following antigen retrieval with Target retrieving solution (DAKO), was used to analyse T/B cell segregation as previously reported . In addiBased on the results of the CD21 staining and the presence of G2/G3 aggregates, samples were qualitatively classified as either diffuse, aggregate/CD21−, or aggregate/CD21+ as previously described . In ordeStaining for AID was performed, as previously reported , on sequT (Threshold Cycle) Method. cDNA from peripheral blood mononuclear cells sorted for CD3 by fluorescence activated cell sorting (FACS) were used as a negative control for AID and CD21L, whereas cDNA from lymph node was used as a positive control.Synovial samples were obtained from an additional 25 patients with RA (the same extra 25 mentioned above). Each specimen was divided into 2 parts; one was formalin fixed and paraffin embedded for immunohistology and the second was stored in a 10:1 v:v of RNA-later (Ambion) at −80 °C for RNA extraction and QT-PCR analysis. Histological characterization of the RA tissue was carried out as described above. Total RNA was extracted from the remaining portion of synovial tissue, using the RNeasy Mini Kit (Qiagen), with on column DNase I digestion to avoid genomic DNA contamination. cDNA was generated from 1 μg of RNA using the Thermoscript RT-PCR System for First-Strand cDNA Synthesis (Invitrogen). QT-PCR was performed to detect mRNA expression levels of AID and CD21L together with CXCL13, LTβ, BAFF and APRIL using specific primers and probes see . The RT-2, and 5 mM DTT for 2 h at 50 °C. The enzyme was inactivated by adding 2% SDS and heating at 100 °C for 3 min and removed by serial spinning and washing with 0.01 M sterile bicarbonate buffer in a 100 kDa Amicon filter device (Millipore). CFb was then biotinylated using NHS-LC biotin (Pierce Biotechnology). In addition, an aliquot of unmodified Fb of the same concentration as the CFb was biotinylated and used as a negative control for all staining experiments.Citrullinated Fb (CFb) was generated as previously described with minor modifications ,44. BrieTo confirm effective citrullination and the specificity of CFb for ACPA Abs, two aliquots of CFb and uncitrullinated Fb were first separated by SDS-PAGE on 7.5% polyacrylamide gels. Proteins were then electrotransferred onto Hybond-C extra reinforced nitrocellulose membranes (Amersham). Membrane strips were probed with human sera (1:100) pooled from three ACPA+ patients with RA and three ACPA– patients with RA. A goat anti-human IgG peroxidase (Sigma) was used for detection of the primary Ab. Peroxidase activity was visualized using ECL Western blotting reagents (Amersham) following the manufacturer's instructions.To detect and characterise ACPA-producing cells in the RA synovium, sequentially cut 5μm paraffin and/or frozen sections of five RA synovial samples representative of different degrees of histological organization  were chosen for analysis. ACPA-producing cells and B cell follicles were identified using double immunofluorescence with biotinylated CFb followed by incubation with streptavidin ALEXA-555 and CD20 followed by rabbit anti-mouse ALEXA-488 with subsequent DAPI counterstaining . AID+ FDA total of 56 samples of human synovium from six patients with RA undergoing arthroplasty  were transplanted subcutaneously into Beige SCID-17 mice as previously described ,46. Each1 ALEXA-488 and CD20 followed by goat anti-mouse IgG2a ALEXA-594.Animals were humanely killed at 4 wk and the grafts divided into two parts; one part was paraffin embedded for later histological examination and one part was stored in 10:1 v:v of RNA-later (Ambion) at −80 °C for QT-PCR. Grafts were examined macroscopically and histologically to confirm viability. In addition, transplanted synovial tissues were characterised by staining for CD21 and AID to identify persisting AID+ FDC networks. Proliferating B cells were identified by performing double immunofluorescence for Ki67 followed by goat anti-mouse IgGFinally, mice underwent a terminal bleed, and serum was collected and stored at −20 °C for subsequent ACPA analysis.RNA was extracted from each transplant, and cDNA was generated as described above. QT-PCR was performed to measure mRNA expression levels of AID, CD21L, APRIL, BAFF, TNFα, LTβ, and CXCL13 see .Detection of Iγ-Cμ circular transcripts was performed on the same cDNA. Circular transcripts were detected following 30 cycles of RT-PCR amplification using the reverse primer Cμ (5′-GTTGCCGTTGGGGTGCTGGAC-3′) together with the forward primer Iγ (5′-GGGCTTCCAAGCCAACAGGGCAGGACA-3′) recognizing both Iγ1 and Iγ2 (the RT-PCR product was 557 bp) as previously reported . Before ACPA were detected in both mouse and human sera using a commercially available anti-cyclic-citrullinated antibody (anti-CCP2) ELISA kit (Axis Shield), following the manufacturer's instructions. A positive result was taken as > 5 U/l.2 test with Yates' correction when required or Fisher's exact test when appropriate were used to evaluate associations of qualitative variables in the different groups. Spearman's rank correlation was performed to correlate expression levels of AID and CD21L mRNA in RA synovial grafts. All the statistical analyses were performed using GraphPad Prism version 3.03 for Windows (GraphPad Software). A p-value of < 0.05 was considered statistically significant.Differences in quantitative variables were analysed by the Mann Whitney U test when comparing two groups, and by the Kruskal-Wallis with Dunn's post test when comparing multiple groups. χIn order to investigate whether AID was expressed within the rheumatoid synovial membrane and its potential association with specific patterns of rheumatoid synovitis, we first performed an IHC analysis on sequential sections of synovial tissue in 24 patients with RA. We analysed AID expression in relation to the degree of lymphoid organization of the inflammatory infiltrate, including T/B cell segregation and FDC network formation.As shown in a representative example in p < 0.0002). Amplicons of the expected length , aggregate pattern AID−/CD21− in seven (28%) patients, and diffuse infiltrate negative for both AID and CD21 in 11 patients (44%). On the other hand, when the same samples were analysed by QT-PCR, we demonstrated a higher prevalence of RA synovial tissues expressing AID and CD21L mRNA . Notably, the increased proportion of RA synovial tissues expressing AID and CD21L mRNA was exclusively characterised by an aggregate and not a diffuse pattern, confirming that lymphoid structures are required for the expression of both AID and CD21L mRNA in the RA synovial membrane.As synovial tissues containing sites of ectopic lymphoneogenesis have been demonstrated to be closely associated with increased expression levels of CXCL13 and LTβ mRNA , we thenIn addition to AID+ B cells within FDC+ networks, a second set of cells within the synovial membrane expressed AID outside this compartment. These cells were characterised by a large cytoplasm, often with a dendritic morphology, and were localized in close association with neighbouring T cells exclusively in CD21+ aggregates A and 3B.We next investigated whether AID+/FDC+ GC-like structures within the rheumatoid synovial membrane were associated with the in situ production of ACPA.Biotinylated citrullinated fibrinogen (CFb) was produced as previously reported  and testACPA+ cells were localised around AID+/CD21+ aggregates E and 4F,The histomorphological analysis described above suggested that ectopic AID+/CD21+ aggregates are capable of sustaining in situ ACPA production in the RA synovium. We next examined a) the direct functional relationship between the synovial expression of AID and CD21L and the production of ACPA and b) whether ectopic lymphoid aggregates within RA synovium maintain functionality independently from incoming immune cells from the periphery. To this end, we generated Hu-RA SCID mice chimeras by implanting fragments of RA synovial tissue into these animals A and 5B,Histomorphology and immunohistological analysis of paraffin-embedded sections of transplanted RA synovium demonstrated viable grafts with residual inflammatory infiltrates C, often To evaluate whether the sustained expression of AID and CD21L mRNA in RA synovial grafts was associated with dynamic gene expression regulating ectopic lymphoneogenesis, we analysed mRNA expression levels of CXCL13, LTβ, TNFα, APRIL, and BAFF. As shown in In order to confirm that AID expression in RA synovial grafts was functional, evidence of ongoing CSR was sought by analysing for the expression of circular transcripts. Circular transcripts are known to be specifically associated with AID expression and exclusively detectable in B cells undergoing CSR, but not by plasma cells, which have already undergone CSR . By perfn = 20) obtained from the same joint of the same patient with RA were stratified according to the presence or absence of AID as determined by QT-PCR Click here for additional data file.Alternative Language Abstract S2(32 KB DOC)Click here for additional data file.Alternative Language Abstract S3(34 KB DOC)Click here for additional data file.


Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy.Mutations in Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin , as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients  and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress.In this study, we investigated the impact of Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.Our results indicate that  Parkin gene (MIM 602544) are the most common known cause of early-onset Parkinson disease , accounting for up to 77% of the cases with an age of onset <30 years Parkin encodes a 465-amino-acid protein with a modular structure Mutations in the Parkin loss-of-function mice presented with reduced expression of mitochondrial function- and oxidative stress-related proteins, decreased mitochondrial respiratory capacity and increased oxidative damage In addition to Parkin's roles as E3 ligase and neuroprotectant, it has been reported to be involved in mitochondrial function Parkin mutations consistently showed reduced mitochondrial complex I activity, coinciding with reduced ATP synthesis rates Investigation of mitochondrial function in human samples supports the findings from animal studies. Functional assays in leukocytes as well as fibroblasts of patients with homozygous or compound-heterozygous Parkin causes swollen mitochondria in Drosophila indirect flight muscles Parkin mutation have been used to investigate mitochondrial morphology and revealed a greater degree of mitochondrial branching in the patients than in controls Another intriguing finding is that Parkin is involved in the regulation of mitochondrial morphology. The knockdown of Drosophila, terminally damaged mitochondria are labeled for degradation by ubiquitylation of mitofusion (mfn) Recently, several publications linked Parkin and mitophagy in different cellular models Parkin-mutant fibroblasts for changes in mitochondrial function and morphology.Most of the above-mentioned data explaining Parkin's function with respect to PD were either obtained in animal models or in small sets of human cellular samples. Here, we evaluated a larger sample of The study was approved by the ethics committee of the University of Lübeck and all participants gave written, informed consent.Parkin alleles and five age-matched controls (mean age±STD: 51.8±11.5 years) without mutations in known PD genes. Phenotypic and genotypic data are summarized in Skin biopsies were obtained from 11 individuals including six affected cases (mean age ± STD: 56.2±13.3 years) with two mutant 2. In all assays, fibroblast passage numbers were matched (<15).Fibroblasts were cultured in high glucose Dulbecco's Modified Eagle's Medium supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin  at 37°C, 5% COTo induce oxidative stress, cells were treated with 2 mM paraquat for 24 h .c Protein Assay Kit  following the manufacturer's instructions. Fibroblasts were harvested and diluted with cell suspension buffer  to a concentration of 1 mg protein per ml. ATP synthesis was initiated by the addition of 250 µl of the cell suspension to 750 µl of substrate buffer . Cells were incubated at 37°C for 10 min. At 0 and 10 min, 50 µl aliquots of the reaction mixture were withdrawn, quenched in 450 µl of boiling 100 mmol/l Tris-HCl, 4 mmol/l EDTA pH 7.75 for 2 min and further diluted 1/10 in the quenching buffer. The quantity of ATP was measured in a luminometer  with the ATP Bioluminescence Assay Kit  following the manufacturer's instructions. Cellular ATP levels were quantified in intact cells as described Cellular ATP synthesis rates were determined according to a published protocol To investigate respiratory chain function, mitochondria were isolated as published The mitochondrial membrane potential was analyzed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide  All measurements were performed in duplicate and in at least three independent runs per sample on a microplate reader .Protein carbonyl levels were measured with the OxyBlot kit , according to the manufacturer's recommendations. Carbonyl groups in the protein side chains were derivatized to 2, 4-dinitrophenylhydrazine (DNP). Western blot analysis was performed with an antibody against DNP. Equal loading was assessed using an antibody against mouse polyclonal anti-β-actin . The experiment was performed three times and a representative blot was analyzed densitometrically with TotalLab software .The mitochondrial network in fibroblasts was stained with an anti-GRP75 antibody  in combination with the zenon immunolabelling kit  according to manufacturer's protocol.m2]/[4πAm]), where Pm is the length of the mitochondrial outline and Am is the area of the mitochondrion The mitochondrial network morphology was investigated using a fluorescence microscope equipped with an ApoTome and AxioVision software . By means of ImageJ 1.42, raw images were binarized, mitochondrion area and outline were measured and the form factor was calculated  reduction of both parameters in mutants compared to controls .Parkin-mutant fibroblasts and controls.We next investigated whether the lower ATP synthesis rates and cellular ATP levels in the patient samples were due to a dysfunction of respiratory chain enzymes. Kinetic assays performed in mitochondrial preparations showed no significant differences in complex I activity in patient fibroblasts  compared to controls . Furthermore, we performed an NADH ferricyanide reductase assay, which allows to determine the content of functional complex I We then went on to determine the mitochondrial membrane potential as a central parameter of mitochondrial integrity. Under basal conditions, the membrane potential was similar in patients and controls, whereas under paraquat-induced stress, the mutants showed a significant (p = 0.028) loss .Parkin mutants, we applied an OxyBlot. This technique demonstrated significantly (p = 0.038) higher levels of oxidized proteins in the Parkin-mutant samples than in controls  in the latter group  higher in mutants than in controls .Parkin cause abnormal mitochondrial function and morphology in PD patient fibroblasts.In this study we demonstrate that mutations in Parkin-mutant fibroblasts under basal conditions. This difference between mutants and controls became more pronounced when the cells were exposed to paraquat. In keeping with our findings, a deficiency of the Parkin interaction partner PINK1 has been reported to cause mitochondrial accumulation of calcium in mammalian neurons, resulting in a mitochondrial calcium overload which then stimulates the production of reactive oxygen species (ROS) via NADPH oxidase Oxidative stress is a key element implicated in the pathophysiology of PD, as recently further supported by studies on human skin fibroblasts from PD patients Parkin-mutant fibroblasts, the authors reported that a decrease in ATP production was linked to complex I Parkin mutations compared to controls. By contrast, we saw no significant difference in complex I activity between patient samples and controls. Also the activities of complexes II to IV were not significantly altered in the Parkin-mutant cells. A possible explanation for this discrepancy might be a loss of the electron carrier glutathione or oxidation of ubiquinone due to increased oxidative stress in the patient cells.Furthermore, there is strong evidence that a deficit in respiratory chain function is involved in the pathogenesis of PD Parkin mutants under basal culturing conditions. However, exposure to high levels of ROS caused a significant decline of the membrane potential in these cells. In an earlier study, the membrane potential was found to be decreased in Parkin-mutant fibroblasts already under basal conditions and culturing in glucose depletion medium supplemented with galactose further worsened the situation When quantifying the mitochondrial membrane potential as a central factor of mitochondrial integrity ParkinPINK1Parkin-mutant cells were found to be more prone to enter fusion as reflected by a significant increase in mitochondrial branching in the patient group Parkin mutants and controls under basal conditions but an increase in mitochondrial mass in the former. In the above-mentioned study Parkin-mutant and control cells to a comparable extent. Similarly, in our study, no significant differences in branching between mutants and controls were detected after exposure to paraquat.Recently, Parkin has been shown to act downstream of PINK1 in a common pathway which appears to regulate mitochondrial morphology Drosophila, the initiation of mitophagy depends on ubiquitylation of the fusion factor mfn by parkin. Following mfn ubiquitylation, dysfunctional mitochondria are prevented from re-fusion with functional mitochondria Parkin mutants under stress, one could imagine that mitochondria with disturbed respiratory chain function are no longer separated and eliminated from the general pool but dominate cellular (dys)function. This effect is in keeping with decreased ATP synthesis rates, elevated oxidized protein levels, increased mitochondrial mass and the observed stress-induced loss of mitochondrial membrane potential in the patient fibroblasts investigated here. Furthermore, one would expect that due to impaired Mfn1/2 deactivation/degradation, mitochondria should be less fragmented in Parkin-mutant than in control cells under stress conditions. Since mitochondrial fusion and fission are transient events, dynamic techniques to quantify the degree of mitochondrial branching would be preferable to the method established so far In light of the most recent publications ascribing Parkin a role in promotion of mitophagy Parkin-mutant cells towards survival. The results from our study underline that also non-neuronal cells, such as fibroblasts from patients, allow important insights into the mechanisms underlying PD and, therefore, will be a useful tool to pursue this scientific goal.In the future, further investigations of the relationship between changes in mitochondrial dynamics/turnover and cell death will be necessary to identify potential targets for neuroprotective drugs redirecting


Previous reports indicate an association between autism spectrum disorders (ASD) and disorders of mitochondrial oxidative phosphorylation. One study suggested that children with both diagnoses are clinically indistinguishable from children with idiopathic autism. There are, however, no detailed analyses of the clinical and laboratory findings in a large cohort of these children. Therefore, we undertook a comprehensive review of patients with ASD and a mitochondrial disorder.We reviewed medical records of 25 patients with a primary diagnosis of ASD by DSM-IV-TR criteria, later determined to have enzyme- or mutation-defined mitochondrial electron transport chain (ETC) dysfunction. Twenty-four of 25 patients had one or more major clinical abnormalities uncommon in idiopathic autism. Twenty-one patients had histories of significant non-neurological medical problems. Nineteen patients exhibited constitutional symptoms, especially excessive fatigability. Fifteen patients had abnormal neurological findings. Unusual developmental phenotypes included marked delay in early gross motor milestones (32%) and unusual patterns of regression (40%). Levels of blood lactate, plasma alanine, and serum ALT and/or AST were increased at least once in 76%, 36%, and 52% of patients, respectively. The most common ETC disorders were deficiencies of complex I (64%) and complex III (20%). Two patients had rare mtDNA mutations of likely pathogenicity.Although all patients' initial diagnosis was idiopathic autism, careful clinical and biochemical assessment identified clinical findings that differentiated them from children with idiopathic autism. These and prior data suggest a disturbance of mitochondrial energy production as an underlying pathophysiological mechanism in a subset of individuals with autism. Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication, as well as isolated interests and repetitive or stereotyped behaviors In 1998, Lombard postulated mitochondrial dysfunction as a cause of autism We reviewed medical records of 25 children with a diagnosis of ASD according to DSM-IV-TR criteria Children were referred for genetic and/or metabolic evaluation of autism but not specifically for evaluation for mitochondrial disease. Children with known chromosomal or monogenic disorders were excluded. All patients met criteria for probable or definite mitochondrial disease by both the Modified Walker Criteria and the Mitochondrial Disease Criteria (MDC) Medical records of each patient were independently reviewed by at least two members of the study team. Growth parameters were evaluated using the ABase software and North American pediatric reference values Twenty-three of the 25 children underwent quadriceps muscle biopsies, 11 had skin biopsies, and one had a liver biopsy. Three patients had sibs with muscle biopsies. Muscle ETC determinations were performed at either Horizon Molecular Medicine, LLC, Atlanta Sixteen of 25 children underwent analyses for selected mitochondrial mutations and 11 of 25 patients had mtDNA mutation analysis by dHPLC of the entire mitochondrial genome at Transgenomic, Inc, Omaha Twenty-five patients–13 males and 12 females ages 2–20 years–were included in this analysis; 11 patients met DSM-IV-TR criteria for autistic disorder and 14 met DSM-IV-TR criteria for PDD-NOS. Twenty-one patients had definite mitochondrial disease and four had probable mitochondrial disease by the Modified Walker Criteria. Eleven patients met criteria for definite mitochondrial disease and 14 met criteria for probable disease using the MDC.Twenty-one patients had histories of major non-neurological medical problems, most commonly of the gastrointestinal system, with gastroesophageal reflux affecting nine and constipation affecting eight subjects. Seven patients had structural or functional cardiovascular abnormalities. In addition, 17 patients had excessive fatigability or exercise intolerance and several children had abnormal physical exam findings including six with facial dysmorphism, four with microcephaly, four with macrocephaly, and five with growth retardation.Twelve patients had neurological findings uncommon in ASD apart from microcephaly, including: oculomotor abnormalities (n = 4), sensorineural hearing deficit (n = 3), dysarthria (n = 3), ptosis (n = 2), movement disorders (n = 2), and hypertonia (n = 1). Five patients had seizures with one having medically refractory epilepsy.Eight patients had markedly delayed early motor milestones. Of 14 patients with regression of previously acquired skills, nine had multiple regressions, six regressed at ages older than three years, and seven had regressions occurring with infections or other metabolic stresses. In six, gross motor skills were lost in addition to language skills. In one case, the timing of regression coincided with a recent vaccination.Family history data were notable for likely autosomal recessive inheritance in one patient whose parents were first cousins. The family history suggested mitochondrial inheritance in three patients. Three other patients had mothers with features suggesting mitochondrial disease and four patients had sibs with either enzyme-defined mitochondrial disease and/or a clinical history consistent with mitochondrial disease.Blood lactate was measured in all patients; 19 had at least one increased lactate level, 13 had multiple high levels and 14 had at least one lactate level greater than 3 mmol/L. Of 17 patients who had at least one blood pyruvate determination, nine (53% of those tested) had at least one increased pyruvate level. Plasma amino acids were analyzed for all patients; nine had at least one increased plasma alanine level and four had multiple high alanine levels. The level of AST and/or ALT was increased in 13. One patient had an increased ALT with a normal AST and seven had elevated AST with normal ALT. Eight patients (28%) had increased serum CK levels. Ten patients (42% of those tested) had urinary organic acid abnormalities indicative of mitochondrial dysfunction. The fibroblast lactate/pyruvate ratio was increased for three patients.Twenty-one patients underwent cranial MRI studies; five also had cranial MRS. Ten children had abnormalities on MRI (48% of those imaged), without a single finding predominating, while two patients had increased lactate on MRS (40% of those imaged).The most common histological abnormalities in muscle were atrophic or regenerating fibers (n = 8), reduced cytochrome oxidase (COX) staining (n = 6), variation in fiber size (n = 5), and increased myofiber lipid (n = 5). The most common ultrastructural abnormalities were abnormal mitochondrial morphology (n = 4) and increased number of mitochondria (n = 4).Twenty patients had deficient activity of a respiratory chain complex on tissue ETC or polarographic analysis; three others had phenotypes and biochemical profiles consistent with mitochondrial disease and a sib with less than 20% activity on muscle ETC determination. Specific abnormalities included complex I defect in 16, complex III defect in five, complex II defect in two, and complex IV defect in one. Two patients had mtDNA mutations of likely pathogenicity and four had mtDNA sequence variants of unclear pathogenicity.The cohort of 25 patients reported here comprises the largest group of individuals with co-occurrence of ASD and defective oxidative phosphorylation reported to date. While previous case reports implicated an association of ASD and mitochondrial dysfunction, it could be argued that this was a chance occurrence in those individuals. Recent epidemiological studies indicate a population prevalence of ASD in about one in 166 children Our results indicate diverse and complex developmental, neurological, and medical phenotypes of persons with mitochondrial autism, nearly all of which differ from those of patients with idiopathic ASD. Although many children with ASD exhibit some degree of hypotonia, most attain their early gross motor milestones on time Recently, there has been increased concern regarding a possible causative role of vaccinations in autistic children with an underlying mitochondrial cytopathy In addition to atypical developmental patterns, non-neurological disorders were nearly universal in our patients. Although medical co-morbidities are not uncommon in individuals with ASD, they are not reported to be present with the high frequency noted here Along with medical co-morbidities, an increased frequency of prenatal and perinatal complications has been reported in children with ASD Besides prenatal complications, our data indicate a high frequency of multiple gestation births. The relevance of this finding is unclear but interesting in view of previous controversy about twinning as a risk factor for autism Also dramatically different from the general ASD population, the sex distribution of the subjects in our cohort is approximately 1∶1. Primary ASD is 3–6 times more common in males than females and this ratio is even higher in PDD-NOS From a biochemical perspective, patients' blood and urine laboratory data revealed marked interindividual variation that did not appear to correspond with specific ETC defects or clinical phenotypes. Significant intraindividual variation was also noted for determinations of blood lactate and pyruvate levels, plasma alanine levels, serum transaminases and CPK, and urinary organic acids, with intermittent normal levels in most cases. The histological and ultrastructural abnormalities noted on muscle biopsies were also varied amongst the subjects of this cohort and similar to results noted in other cohorts of children with mitochondrial electron transport chain disorders in vitro does not prove a genetic defect of oxidative phosphorylation because ETC deficiencies can be secondary to other conditions For most individuals with defects of oxidative phosphorylation, the diagnosis is made through ETC determination but an underlying nuclear or mitochondrial mutation usually cannot be identified ETC complex I deficiency was the most prevalent enzyme defect, affecting 64% of our patients, followed by complex III deficiency, affecting 20%. It should be noted, however, that we cannot exclude the possibility that some patients might have biochemically mild forms of multiple ETC deficiencies. The predominance of ETC complex I deficiency is not unexpected and has been noted in cohorts of non-autistic patients with mitochondrial cytopathies Ileu, reduces 3′ tRNAse processing efficiency in vitro functional studies preclude assignment of probable pathogenicity at this time. For all three patients with these mutations there was intrafamilial phenotypic heterogeneity, as well as differences from phenotypes reported in the literature. Undetected heteroplasmy could explain these differences, but intrafamilial phenotypic variation with homoplasmic mitochondrial mutations has been reported and reasonable explanations proposed Of patients who underwent mitochondrial genomic analysis, two had rare homoplasmic DNA sequence variants of likely pathogenicity: mtDNA 3397A>G and 4295A>G. mtDNA 3397A>G, which replaces methionine with valine in a highly conserved region of the ND1 subunit of complex I, has been implicated in various neurological phenotypes and other conditions Overall, our results demonstrate substantial clinical heterogeneity of individuals with co-occurring autism and defects of mitochondrial oxidative phosphorylation, nearly all of whom we found to be clinically distinct from children with idiopathic autism. The data do not exclude the possibility of persons with isolated autism having a disorder of oxidative phosphorylation–in fact, one of our patients did not have any major clinical features that distinguished her from typical autism. In addition, it is possible, if not likely, that a still broader clinical, biochemical and genetic spectrum of mitochondrial autism exists.Finally, data from multiple disciplines, especially research in developmental neurobiology and genetics, point to several underlying pathophysiological mechanisms in autism, including altered neurite morphology, synaptogenesis and cell migration due to abnormalities in distinct ensembles of proteins and pathways Table S1ASD Diagnosis and Mitochondrial Disease Criteria. PDD-NOS = Pervasive developmental disorder–not otherwise specified, MDC = mitochondrial disease criteria(0.07 MB DOC)Click here for additional data file.Table S2Pertinent Medical History. GERD = gastroesophageal reflux disease, POTS = postural orthostatic tachycardia syndrome, RBBB = right bundle branch block, EF = ejection fraction, LVH = left ventricular hypertrophy(0.05 MB DOC)Click here for additional data file.Table S3Regression History. Blank cells indicate an absence of regression of the type listed in the column(0.05 MB DOC)Click here for additional data file.Table S4Biochemical Data. L:P = lactate to pyruvate ratio, DCA = dicarboxylic acids, EMA = ethylmalonate, 3-MG = 3-methylglutaconate * Value is listed only if level was higher than the upper limit of the reference interval † Patient had one high level but exact value is unknown(0.06 MB DOC)Click here for additional data file.Table S5Neuroimaging. Blank cells indicate that patient did not undergo cranial MRI(0.04 MB DOC)Click here for additional data file.Table S6Enzymology and mtDNA Testing 1 Testing performed at CIDEM Lab, Cleveland 2 Testing performed at Horizon Molecular Medicine LLC, Atlanta A: selected point mutations screened B: mtDNA sequencing C: whole mitochondrial genome scan by dHPLC P: mutation of probable pathogenicity U: mutation of unclear pathogenicity OXPHOS: polarographic determination of mitochondrial oxidative phosphorylation reactions * This patient does not have mitochondrial sequence changes indicative of haplogroup K †ETC complex III, when measured separately, was normal in these patients(0.06 MB DOC)Click here for additional data file.


Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer , smokers without lung cancer , and never smokers . Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure. Approximately 1.3 billion people smoke cigarettes worldwide, contributing to almost 5 million preventable deaths per year et alPrevious studies have demonstrated that cigarette smoke exposure creates a “field of injury” in airway epithelial cells  gene expression and lung cancer status, (2) SNP genotype and gene expression, and (3) SNP genotype and lung cancer status.Human genetic variability in the response to environmental exposure is generally accepted as an important determinant in susceptibility to cancer et alUsing the approach described by Spira With the aim of identifying potential functional SNPs and/or haplotypes in antioxidant response pathways associated with smoking-induced lung cancer, we used a three-part approach to analyze the associations between: 1) gene expression and lung cancer status; 2) gene expression and genotype of SNPs selected by several bioinformatics strategies, and 3) SNP genotypes and lung cancer status. The overall workflow of our approach is outlined in Bronchial airway epithelial cells were obtained by flexible bronchoscopy from 8 healthy never-smokers (NS) and from smokers enrolled in a diagnostic study for clinical suspicion of lung cancer including 20 smokers with lung cancer (SC), and 24 smokers without lung cancer (SNC) . These s2-expression values, and statistical values are included in the To examine the differential responses to the effect of cigarette smoking on the bronchial airway transcriptome, we used profiles from never-smokers as baseline and compared the average log2-expression values of SC or SNC with that of NS by t-test, followed by multiple testing correction  We also used IPA to test for enriched canonical pathways using lists of over-expressed or under-expressed probe-sets and determining the relative weight of identified pathways in the different phenotype groups. For the 210 over-expressed probe-sets between SNC and NS, six pathways were significant , includiet alet alin vivo repressed ARE-dependent gene expression and modulated response to oxidative stress. That is, NRF1 was observed to be anti-correlated with expression of NRF2 pathway genes. MAFG was lower in SC compared to NS or SNC . No significant changes were found in NRF3, KEAP1, and BACH1 mRNA  in the upstream regions). As a result, we examined this group of genes more closely. Under oxidative or electrophilic stress, NRF2 is released from its interaction with KEAP1 and translocates to the nucleus, where it heterodimerizes with small MAF proteins such as MAFG, and then binds ARE sequences upstream of NRF2 target genes CH1 mRNA . As expeCH1 mRNA . NRF2 taWe re-examined a larger, previously published dataset In order to examine if genetic variation was contributing to airway gene expression differences, we used a bioinformatics strategy to identify SNPs in potential NRF2 binding sites Expression analysis suggested that MAFG levels could potentially be rate-limiting in the expression of antioxidant response genes. To better evaluate if sequence variability in the MAFG gene affected gene expression, we sequenced a 16.5-kb genomic region  in our study subjects. This region was from 5000-nt upstream of the transcription start site to 2000-nt downstream of MAFG's transcription end site, covering introns, exons and untranslated regions. We discovered 33 SNPs in this region, including 1 coding SNP, 16 SNPs in 3′ UTR, and 3 SNPs in introns, and 13 SNPs in upstream. The location, allele frequency and linkage disequilibrium (LD) is included in 2-transformed gene expression values and genotypes of SNPs that were near each gene . Statistical significance was evaluated using 10,000 permutations of expression values relative to the genotypes as previously described by Stranger et alp value threshold of 0.05. In 44 smokers, from among 338 detectable probe-sets (213 genes) nearby our selected 312 SNPs, we found significant association between 26 SNPs and 29 probe-sets . We also tested the association between newly identified SNPs in MAFG and MAFG gene expression. The promoter SNP at chr17:77482956  was associated with MAFG expression (p_corrected  = 0.0038).We performed linear regression between normalized logAlthough our focus was the association between genotype and expression in all smokers in the study, we also examined the difference in associations between SC and SNC groups. This type of exploratory analysis could reveal SNP effects, that might be related to differential susceptibility in smokers. An association was found between expression and a putative ARE SNP rs3753660 at -199-nt of epoxide hydrolase 1 (EPHX1) gene . The ass2 = 0.28), were independently associated with expression  in the glutamate-cysteine ligase catalytic subunit (GCLC) gene that were not in LD  in the antioxidant response pathway that were associated with cancer status of smokers at a corrected p-value < = 0.05. Notably, the MAFG probe-set 204970_s_at was associated with “without cancer” status (p = 0.0014) . As outlin vitro cell culture et al identified gene expression profiles in cytologically normal large-airway epithelial cells that can serve as a diagnostic biomarker for lung cancer There is an important heritable component to lung cancer NRF2 is activated by oxidative stress and translocates to the nucleus where it heterodimerizes with small MAF proteins to form a transactivation complex that binds to specific DNA regions termed antioxidant response elements (ARE) cis-acting effects for putative regulatory SNPs on genes affected by cigarette smoking and these could impact susceptibility of the airway to smoking-related diseases through various mechanisms, including metabolism of carcinogens. For example, members of the aldo-keto reductase (AKR) superfamily, AKR1C1, AKR1C2, and AKR1C3, catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and/or NADPH as cofactors. Polymorphisms of AKR1C3 have been implicated in susceptibility to various types of cancer, including lung cancer To explore how genetic variation in other NRF2 pathway genes might contribute to smoking-induced lung cancer disease susceptibility, we tested the association of many genotypes with both expression and/or group phenotype. We observed possible Disease-association studies, both candidate gene-based and genome-wide association studies (GWAS), have identified genetic variants that associate with both monogenic and complex diseases like lung cancer. Recently several genomic regions that may affect nicotine metabolism or dependency in lung cancer patients were identified by GWAS with very high statistical significance The approach we have used integrates information about gene expression in target tissue, variation in transcription factor binding sites and genotype frequency among cancer status groups in order to identify biologically-plausible functional polymorphisms. It could be generally useful for identifying SNPs that contribute to disease risk through their impact on gene expression. While the present study is limited by statistical power, applying this method to larger studies of exposed-tissue samples from clinically characterized patients may reveal useful expression-based and/or genetic biomarkers and provide a basis for prevention efforts.Previously we recruited a group of healthy never smokers  to undergo bronchoscopy at Boston University Medical Center The study was approved by the Institutional Review Boards of all medical centers, and all participants provided written informed consent.Clinical subjects in the latter cohort were classified as either having lung cancer if their bronchoscopy or subsequent lung biopsy yielded lung tumor cells or not having lung cancer if the bronchoscopy or subsequent lung biopsy yielded a non-lung-cancer pathology . Bronchial airway epithelial cells were obtained from the uninvolved right mainstem bronchus with an endoscopic cytobrush . RNA was extracted and its integrity and epithelial cell content was confirmed as described previously Individuals with final diagnoses as of July 2008 and with blood genomic DNA for genotyping were included in this study, consisting of 20 smokers with lung cancer (SC), 24 smokers without lung cancer (SNC), and 8 never-smokers (NS) . We obseApproximately eight micrograms of total RNA was processed, labeled and hybridized to Affymetrix HG-U133A GeneChips containing 22,283 probe-sets as described previously All microarray data have been submitted to the Gene Expression Omnibus (GEO) under accession number GSE 19027.a priori defined set of genes were enriched at the top or bottom of a list of genes ordered on the basis of expression difference between two biological states (e.g. phenotypes). It takes normalized array intensity values, rank genes according to score  between phenotype groups. It then walks through the ranked list of genes to calculate Enrichment Score (ES) for a gene set. In this study, we focused on the canonical pathways, i.e.: the c2.cp library of “molecular signature database” of GSEA software (v2.0.4), which included 639 curated gene sets  from online pathway databases, publications in PubMed, and knowledge of domain experts. In addition, we tested a set of “antioxidant response element genes”, which included 37 human genes that have bona fide NRF2 binding sites (or antioxidant response elements), curated by mining PubMed database GSEA is a computational method http://www.ingenuity.com). Genes from the data set that met the FDR cutoff (q≤0.1) and were associated with biological functions in the Ingenuity Pathways Knowledge Base were considered for analysis. Fisher's exact test was used to determine the probability that each biological function assigned to that data set was due to chance alone.IPA is software for identifying pathways most relevant to experimental datasets  and assayed using SYBR green (Applied Biosystems). For real time analysis we performed 40 PCR cycles using 15 seconds 95°C melting temperature and 1 minute 60°C annealing/extension temperature per cycle and measured fluorescence intensity with an ABI 7900HT and calculated initial fluorescence  of each amplified sample using the method described by Peirson and colleagues . We normalized all target values with ACTB mRNA values. Experiments were performed in triplicate; PCRs were carried out in triplicate and values are reported as mean ± standard error of the mean (SEM).We silenced MAFG in A549 cells by transiently transfecting small interfering RNA (siRNA) targeted to MAFG mRNA. Cells at 90% confluence were transfected with 0.4 µM siRNA  in the presence of 4.7 µl Lipofectamine 2000 transfection reagent (Invitrogen) for each well of a 6-well plate. We incubated transfected cells in antibiotic-free Ham's F12K without serum for 24 h before cell lysate collection. RNA was collected using RNeasy kit (Qiagen), including DNase treatment, and reverse transcribed using Superscript II cDNA synthesis kit (Invitrogen), following manufacturer's instructions. NQO1, GCLC, TXNRD1, MAFG, and ACTB (beta actin) gene expression levels were measured using TaqMan assays (Applied Biosystems). For SLC7A11, we designed expression primers using Primer3  . The SNPs were detected by PolyPhred software developed by University of Washington.2 threshold of 0.8, minor allele frequency threshold of 0.1 was used.Tag SNPs were selected based on Hapmap genotyping data on 60 unrelated CEU individuals (U.S. Utah residents with ancestry from northern and western Europe), using the Tagger software SNPs in putative NRF2 binding sites (or AREs) were selected using an approach developed by our group Utilizing Illumina's Assay Design Tool, we designed Illumina GoldenGate assays for candidate SNPs . DNA samples derived from whole blood were subjected to whole genome amplification prior to genotyping. Post-amplification DNA products were cleaned and genotyped according to the manufacturer's protocol on an Illumina BeadStation 500G GoldenGate genotyping platform. Genotypes were assigned using Illumina's Beadstudio v3.0 Genotyping software with GenTrain scores >0.25. A total of 450 SNPs, including 342 SNPs located in putative binding sites of transcription factor NRF2 and 108 tag SNPs for 16 known ARE genes were placed on Illumina Golden Gate arrays and used to genotype DNA from our study subjects.http://pngu.mgh.harvard.edu/purcell/plink/.A linear regression model was used to evaluate the association between log2-transformed expression values of a probe-set and genotypes of a SNP as previously described http://discover.nci.nih.gov/gominer/), developed by National Cancer Institute, Bethesda, Maryland All other statistical analyses were accomplished using the R statistical software package (v2.8.0) and the SAS JMP statistical discovery software . Gene Ontology analysis was done through the GoMiner web interface Click here for additional data file.


PRF1), a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction.Differential expression of perforin  or DNA extracted from peripheral blood mononuclear cells (PBMC DNA) from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18%) to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation  in the distal enhancer region (CpG sites 1–10), a variable methylation (range 49%–83%) in the methylation sensitive region (CpG sites 11–17), and a progressively declining methylation level (range 12%–80%) in the proximal promoter region (CpG sites 18–32) of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA.We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune function.This reproducible, site-specific and quantitative method for methylation determination of  PRF1), a major immune surveillance gene, is a candidate gene for the identification of methylation markers, and for investigating the pathophysiology of a number of chronic diseases with suspected immune dysfunction [PRF1 promoter methylation used bisulfite sequencing of cloned PCR products [PRF1. Pyrosequencing determines the ratio of C/T base changes after bisulfite treatment, reflecting the proportion of unmethylated and methylated cytosines at each CpG site in the original sequence [Perforin PRF, a majorfunction . Earlierproducts . Cloningsequence .Several factors like sample collection, bisulfite treatment, and PCR can affect the reproducibility of methylation levels determined by pyrosequencing. Blood is a non-invasive source of sample for molecular epidemiologic studies. Usually, blood is drawn into blood collection tubes followed by separation of peripheral blood mononuclear cells (PBMCs) to extract DNA for molecular genetic studies including methylation. Recently, to minimize variability in primary blood samples from multicenter studies and to enable transport and storage, blood is drawn into specialized tubes like PAXgene blood DNA tubes containing additives for cell lysis. These PAXgene blood DNA tubes are then transported to laboratories where DNA is extracted directly from the whole blood without PBMC separation. DNA extracted from PAXgene blood DNA tubes thus represents the contribution of all blood cell types and have been successfully used for genotyping. However, the impact of these blood collection procedures for DNA (whole blood vs PBMC DNA) on methylation levels has not been reported.PRF1. The assay is highly reproducible with respect to bisulfite treatment, and PCR. Examination of methylation levels in normal subjects showed that methylation levels differ significantly between DNAs extracted from PBMCs and whole blood for all 32 CpG sites except two, although the methylation pattern remained similar over the 1.4 kb promoter region. These results show that site-specific assessment of CpG methylation in PRF1 can be reproducibly assessed by pyrosequencing without cloning, and that PBMC DNA may be better suited than whole blood DNA for molecular epidemiologic studies on methylation levels of genes associated with immune dysfunction.In this study, we developed a pyrosequencing based methylation assay for a total of 32 out of 34 CpG sites in the promoter region of PRF1 promoter. Similarly, both forward and reverse sequencing designs were employed with 5'biotinylation of the reverse or forward PCR primer respectively. All primers were synthesized at the Biotechnology Core Facility, Centers for Disease Control & Prevention, Atlanta, GA.Bisulfite converted sequences were simulated by a previously reported Microsoft Word bisulfite macro  followedBlood drawn in PAXgene blood DNA tubes  for DNA extraction were obtained from 20 subjects who took part in the Chronic Fatigue Syndrome Study at the Centers for Disease Control and Prevention, Atlanta, GA. From five of these subjects classified as normal, blood was also drawn in BD Vacutainer CPT tubes  followed by separation of PBMC for DNA extraction. DNA from PAXgene blood tubes was extracted using PAXgene blood DNA kit (Qiagen) whereas DNA from PBMCs was extracted using Roche DNA isolation kit for mammalian blood . DNA was quantified using NanoDrop ND-100 Spectrophotometer  prior to bisulfite treatment. DNA (200 ng/reaction) was bisulfite treated using the EZ DNA Methylation Kit , according to the manufacturer's instructions. Bisulfite treated DNA (BST-DNA) was eluted twice in 10 μl of the manufacturer's M-Elution Buffer , and was stored in aliquots at -20°C until use.2, 0.2 mM dNTP, 0.05 U/μl Platinum Taq DNA polymerase  and 0.2 μM each of forward and reverse primers (Table Each 100 μl PCR contained 1.5 mM MgClCDNK2A [Pyrosequencing was performed according to the manufacturer's instructions. The PyroGold kit was used in conjunction with the PSQ 96MA instrument (Biotage), and each pyrosequencing reaction used 20 μl of PCR product. Biotinylated single strand DNA was separated by immobilizing the PCR product on streptavidin coated Sepharose High Performance beads  and strand separated by denaturation with 0.2 N NaOH. After denaturation, the biotinylated strand was annealed to sequencing primer , and subjected to sequencing using an automatically generated nucleotide dispensation order for the "sequence to analyze" corresponding to each reaction Table . PyrograCDNK2A .The impact of bisulfite treatment on assay reproducibility was tested using the PCR and pyrosequencing conditions for CpG sites 18–21 in amplicon G with whole blood DNA from 20 subjects. The same DNA was bisulfite treated on three separate days. Each day, 20 individual bisulfite reactions were set up with 200 ng DNA/reaction. BST-DNA from each of the three days was simultaneously subjected to PCR in the same thermocycler to eliminate PCR-to-PCR variability. All PCR products were used for pyrosequencing in duplicate, giving a mean of 6 pyrogram readings per CpG site for all three sets of BST-DNA per subject. Day-to-day variation in bisulfite reaction was expressed as coefficient of variation (CV) calculated as the percentage of standard deviation to its meanThe impact of PCR-to-PCR variability, expressed in terms of CV, was also tested for CpG sites 18–21 in amplicon G on three different days using the same BST-DNA. Touchdown PCR reproducibility was done with whole blood DNA from 10 subjects. Each PCR product was subjected to pyrosequencing in duplicate, yielding a mean of 6 values per CpG site for PCR from all three days per subject.PRF1 promoter for 32 CpG sites. This pilot study also examined the impact of DNA from whole blood or PBMC on PRF1 methylation profile in normal subjects. For each subject, two separate bisulfite reactions were set up, followed by corresponding PCR and pyrosequencing reactions yielding a mean of 2 values per CpG site. Both whole blood and PBMC DNAs were treated similarly to generate the PRF1 methylation profiles.A small pilot study consisting of five normal subjects was conducted to evaluate the robustness of the optimized conditions to determine the methylation level/pattern in the PRF1 promoter spans about 1.4 kb (-1411 to +1) upstream of the transcriptional start site and harbors a number of transcription factor binding sites [PRF1 promoter, 10 are in the distal promoter region containing enhancer elements (CpG sites 1–10), 17 are in the proximal promoter region harboring repetitive elements (CpG sites 18–34), and seven (CpG sites 11–17) are in the methylation sensitive region (MSR) located between the distal and proximal promoter regions . On the other hand, touchdown PCR consistently generated specific products of expected size with all amplicons except amplicon F, for which PCR with single annealing temperature consistently resulted in specific product than touchdown PCR. Thus, we used touchdown PCR to generate all amplicons, except amplicon F which was generated with PCR following single annealing temperature. Pyrosequencing reactions for all CpG sites were successful except CpG sites 33 and 34 in amplicon K, although amplicon K was successfully generated by the touchdown PCR.Variability due to bisulfite treatment was tested for four CpG sites sites 18–21) in amplicon G using whole blood DNA from 20 individual subjects. Variability due to bisulfite treatment was found to be minimal for all four CpG sites in amplicon G,  in the distal enhancer region covering CpG sites 1–10  that was comparable to the minimal reaction-to-reaction variation in bisulfite treatment as determined by fluorescent probe based quantitative version of methylation-sensitive PCR  [Pyrosequencing assay was designed for all 34 CpG sites in the ion CV 2.% that wat assay) , but thePRF1 methylation pattern identified using both whole blood and PBMC DNAs from normal subjects appeared similar throughout the promoter region in terms of extremely high methylation in distal enhancer region, variable methylation in the MSR, and progressively declining methylation in the proximal promoter region. Except CpG sites 5 and 15, the absolute levels of methylation at each of these CpG sites, however, differed between whole blood and PBMC DNAs, with higher methylation levels obtained using whole blood DNA. We have not investigated the factors that contribute to this similar pattern but different levels in methylation along the PRF1 promoter between whole blood and PBMC DNAs but it is likely to be related to differences in cell types between these samples from the same subject. In a previous study that used bisulfite sequencing followed by cloning, CpG site 14 was found to be the most highly methylated (> 50%) in the MSR of CD4+ T and CD8+ T cells of normal subjects [PRF1 in the various sub-populations of cells in blood from normal and subjects with chronic diseases of immune dysfunction. Further, the study suggests that blood drawn in PAXgene blood DNA tubes may be inappropriate for DNA methylation studies of genes with immune function.In this study, the PRF1 methylation determined by pyrosequencing with other method such as MSP or combined bisulfite restriction analysis since our reproducibility data agree with other reports supporting the reliability of pyrosequencing for quantitative and efficient screening of multiple CpG sites in candidate genes studies [We have not compared the estimates of  studies ,19-21. F studies ,23. A nu studies ,18,23,24PRF1 promoter. In normal subjects, methylation levels along the 1.4 kb promoter varied similarly in DNAs from whole blood and PBMC but differed in absolute methylation levels, suggesting collection of blood in devices like PAXgene blood DNA tube may not be appropriate for methylation studies of immune function related genes. This method can be applied to clinical samples in order to systematically evaluate the significance of multiple CpG sites in perforin which will improve our understanding of the pathophysiology of chronic disorders with alterations in immune function.In conclusion, we have developed a highly reproducible procedure based on pyrosequencing technology for the site-specific and quantitative determination of methylation levels in 32 CpG sites in the The authors declare that they have no competing interests.SN developed the pyrosequencing strategy, carried out the experiments and participated in manuscript writing. VRF contributed to experimental designs, interpretation of results and participated in the manuscript writing. MMK participated in the design, implementation of laboratory experiments, and manuscript writing. MSR conceived of this study and as Principle Investigator participated in all aspects of this study and drafting of this manuscript. All authors have read and approved the manuscript.


Luffa echinata Roxb., commonly known as Bindal in Hindi is used for its hypoglycemic activity in the indigenous system of medicine. No pharmacognostical study on stem is reported in the literature till date; therefore, it was decided to study macroscopical and cytomorphological characters in detail to bring out salient diagnostic features. The stem pieces available in the market are 1.5–17 cm long and 5–8 mm in diameter, showing yellowish-brown to brownish-black surface with longitudinal furrows, fracture is fibrous, and taste is bitter. Mature stem shows single-layered epidermis, seven layers of collenchyma below five ridges but one to two layers of parenchyma in rest of the region beneath the epidermis, continuous wide wavy layer of pericycle composed of three to eight layers of fiber. There are five conjoint bi-collateral open vascular bundles one below each ridge and additional four medullary vascular bundles in the pith each facing furrows. Luffa echinata Roxb. (Cucurbitaceae) is a climber, commonly known in Hindi as Bindal long, narrow with pointed to blunt ends showing wide lumen and simple pits on their walls. Some of the fibers show beak like ends [Figure 2]. Similar to normal vascular bundles, four medullary vascular bundles develop in the pith region each facing the furrows. Some of the parenchymatous cells of phloem show tanniferous content. The xylem fibers are distinctly smaller in size as compared to pericyclic fiber [Figure 1–b4]. Some of these fibers showed notch projection on one or both the ends. The vessels are cylindrical drum shaped with border on their walls ; however, some vessels [Figure 1–d4] show spiral thickening as well as tracheids [Figure 1–f4] vary in shape and show simple pits on their walls and are septed. The xylem parenchyma is also thickened showing simple pits on their walls .The mature stem Figures  and 7 shs Figure : a2. Simr Figure : b1–b4. s Figure : d1–d4 ss Figure : f1–f4 vs Figure : g1, g2.L. echinata Roxb. are given below in microns:Measurement of different cells and tissues of the stem of The stem can be identified by its yellowish-brown to blackish-brown color and longitudinal furrows besides young stem microscopically can be identified by the presence of quadrangular outline, parenchyma followed by a continuous layer of collenchyma, thick and lignified pericyclic fiber, four conjoint bi-collateral vascular bundles and pith with thick-walled and pitted cells. Similarly mature stems can be characterized by its five distinct ridges and furrows, less-thickened pericyclic sclerenchyma, five conjoint bi-collateral vascular bundles facing the ridges, and four medullary bundles facing the furrows and thin-walled and delignified pith cells.


By contrast, WT littermates exhibit Mn2+ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here.The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn The dopamine transporter  acts to terminate dopaminergic neurotransmission through reuptake of dopamine from the synaptic cleft into presynaptic neurons. Dopamine is a key neurotransmitter that can influence cognition, emotion, and movement; and many drugs exert their psychotropic effects via DAT In this work we link the ends of the molecular-to-behavioral spectrum using a panel of magnetic resonance imaging methods to investigate ramifications of DAT KO on mesoscopic scale neuronal circuitry and overall brain morphology in the mouse. By injecting tracer into the prefrontal cortex, we focus on the limbic cortical-ventral striatopallidal circuitry that has been implicated in a number of psychiatric disorders that are thought to involve changes in reward and executive functions mediated by the prefrontal cortex (PFC), including addiction e.g. glutamate, glutamine, lactate, N-acetyl-aspartate, choline, myo-inositol, taurine) that are present in the brain at millimolar or greater concentrations and can indicate disease states 2+ into the PFC reveals active neuronal connections that emanate from the PFC. Computational co-registration of all the 3D images allows the determination of statistical parametric maps (SPM) that identify changes on a voxel-wise basis without the need to define regions of interest. These results thus provide a comprehensive unbiased approach to identification of the sites to which Mn2+ is carried from the PFC injection site. SPM analysis of intensity versus time provides information about how Mn2+ accumulation propagates from the injection site. Tensor based morphometry Magnetic resonance spectroscopy (MRS), manganese enhanced MRI (MEMRI), and diffusion tensor imaging (DTI) data were obtained in DAT KO and WT mice using protocols similar to previous studies with SERT KO mice Inspection of MR images recorded an hour after injection revealed that the center of each injection site fell within a 0.2 mm radius for all 16 animals. The average injection site (± standard deviation) was: x  +0.94±0.07 mm; y (anterior-posterior with reference to Bregma) +1.04±0.14 mm; z  −1.29±0.21 mm. Histological examination of the injection site revealed minimal damage due to the injection procedure , and con3 volume centered in the striatum. Relative concentrations for each metabolite are shown in We determined concentrations of metabolites relative to creatine plus phosphocreatine, which is assumed not to vary among mice or between the two genotypes, by probing the striatum of both genotype groups. All spectra were recorded from a 2 mm2+-induced hyperintensity in the MRI data sets, we performed image alignment and statistical parametric mapping to compare how DAT KO and WT mice differed in the time dependence of Mn2+ accumulation at locations distant from the injection site.Image alignment allows voxel-wise comparisons in both human and small animal experiments For the tensor based morphometry analysis, the structural and isotropic diffusion weighted (iDWI) data sets from DAT KO and WT mice that were obtained prior to any injections provided input. Very few voxels displayed deformation tensor indices that differed between DAT KO and WT mice. At a p<0.001 level of significance no voxels were significantly different in the logmat SPM. Only 0.8% of voxels reached this level of significance in the det SPM.i.e. the propensity of water to diffuse along, rather than perpendicular to, a nerve bundle). TrD is a measure of the orientation-independent mean diffusivity of water in the tissue. Fractional anisotropy and trace of the diffusion tensor are two rotationally-invariant indices derived from the diffusion tensor that provide information about underlying tissue characteristics n vivo MR images then b) calculating the minimum deformation target (MDT) at each time-point, which provides an unbiased average. The MDT images preserve the fidelity of the original images. Anatomical structures are clear and distinct; ventricles, corpus callosum, hippocampus, internal and external capsule are all easily identifiable. To provide an unbiased assessment of the whole brain, we performed statistical parametric mapping to determine the spatial extent of Mn2+ accumulation, detected by Mn+2-induced signal intensity increase, as a function of time after injection. Color-coded volumes denote significant  increases in intensity from one time point to the next. Representative slices from the 3 dimensional data sets are shown in 2+ into the prefrontal cortex . To check that the intensity increase near 4V was not due to a single outlier, we repeated the Student's t-test parametric map analysis 8 times, each time dropping a different single sample from the analysis. The intensity increase near 4V was observed in all the comparisons.The injection site is highlighted (green). This denotes the significantly more intense signal induced by injecting Mnl cortex , 5 and 6l cortex , sagittal cortex , and tral cortex  views, Mt-test comparisons of the 4 hr post-injection image datasets with those recorded at 1 hour reveal widespread changes . In this study we have used a panel of magnetic resonance methods, as well as histological confirmation, to investigate changes brought about by lifetime genetic deletion of the dopamine transporter. This study parallels an earlier examination of the serotonin transporter knockout mouse Neither metabolite concentrations nor anatomical differences were found between DAT KO mice and their WT littermates. MRS detectable metabolite concentrations in the striatum were at similar levels in WT and DAT KO mice. There was thus no evidence that these animals differ from wildtype mice in neuronal viability (indicated by NAA), excitatory and inhibitory system integrity (indicated by Glu and GABA), membrane turnover (indicated by Cho), or glial volume (indicated by Tau) in vivo pre-injection structural MRI and ex vivo iDWI data sets. These assessments also revealed few statistically significant differences between genotypes. Although DAT KO mice are known to have somewhat smaller brains than WT mice , and more basal anterior structures. Thus, WT mice display Mn2+ accumulation reflecting the same connectivity along the cortico-mesolimbic pathway as has been assessed in classical tracing work 2+ movement was truncated at 26 hr post injection. In the DAT KO mice at 26 hr post-injection the hyperintensity distribution includes a substantially smaller subset of the WT distribution and Mn2+ accumulation failed to reach such posterior structures as medial entorhinal cortex, deep mesencephalic nuclei, and subiculum. Thus, deletion of the DAT gene produces alterations in mesocortical circuitry that originate from the PFC.MEMRI revealed major differences in the pattern of connectivity between the prefrontal cortex and distal brain regions between the DAT KO mice and their WT littermates. The complex statistical parametric maps can be assembled into a simplified diagram of the anatomical locations occupied as Mnhe brain . This scThe time-course and spatial extent of the circuitry traced out using MEMRI in the WT littermates of DAT KO mice was substantially different than that observed for the WT littermates of SERT KO mice reported in our previous study, although the reasons for these differences are not clear 2+ is primarily an anterograde trans-synaptic tracer that is transported via fast axonal transport and accumulates intracellularly to levels sufficient to induce increased intensity in T1-weighted MR images at locations distant from the injection site 2+ is an activity dependent tracer that is taken up by active neurons, probably via voltage and/or ligand gated Ca2+ channels 2+ for any particular type of neuron. However, although all active neurons at the site of injection may have taken up the tracer, the initial projections observed in this work are likely to arise from the pyramidal projection neurons of the prefrontal cortex As in all studies employing neuronal tracers, interpretation of the results depends on the properties of the tracer. MnStudies of DAT KO mice have used a wide variety of behavioral, pharmacological, physiological, and ultrastructural measures to suggest that this knockout induces significant adaptive changes at the synaptic, cellular, and molecular levels. These data include region-specific changes in various dopamine receptor densities and altered responses in other neurotransmitter systems 2+ accumulation is seen in the DAT KO mice on the midline bordering the base of the fourth ventricle. Such accumulation could be either a consequence of migration around the brain of Mn2+ leaked at the brain surface during the injection procedure or fast axonal transport through the brain parenchyma. Timing of passage around or through the cerebrum are both consistent with appearance of Mn2+ at this location an hour after its introduction. Within half hour of introduction into the cisterna magna of the rat, india ink tracer distributes as far forward as the olfactory bulbs and along paravascular pathways of the middle cerebral artery and its branches on the superior surface of the brain 2+ accumulation in areas near 4V may indicate active connections from the prefrontal cortex to periventricular areas below 4V in the DAT KO mouse that are not present, at least to the same extent, in their WT littermates. Limited resolution and contrast in the MR images and parametric maps coupled with the existence of many small nuclei in this area preclude definitive identification of the specific structures involved.At the earliest time-point in our series (1 hr post-injection), Mn2+ is transported from the prefrontal cortex to most of the expected areas in both WT and KO mice: caudate putamen (CP), globus pallidus (GP), nucleus accumbens (NAc), thalamus (TH), and substantia nigra (SNr). These areas represent a large subset of the known connections between the neocortex and the basal ganglia of the rodent brain 2+ accumulation between 4 hr and 8 hr in the DAT KO  than that observed in DAT KO mice. When basal activity differences are conflated with connectivity differences, as they are in MEMRI, it is not surprising that somewhat different patterns emerge in the MEMRI brain scans compared to techniques that measure basal metabolism only, such as 18FDG uptake in PET scans. Nonetheless, these imaging results, taken together, indicate that the DAT KO brain is both qualitatively and quantitatively different than the corresponding WT brain, with substantial effects on the reward pathways that may involve several of the monoamine systems.By 26 hr, MnThe DAT KO mouse has been suggested to constitute an animal model of several disorders thought to involve hyperdopaminergic function, including schizophrenia In this study we employed a panel of magnetic resonance methods to probe the effects of lifelong deletion of DAT in a mouse model system. As detected by magnetic resonance methods, no significant changes in metabolite levels or morphology were observed in DAT KO mice. In contrast, MEMRI revealed that DAT KO mice had extensive alterations in the executive portions of reward circuitry that are intimately involved in addiction, as well as other psychiatric conditions. Connections originating in the prefrontal cortex were significantly truncated in the DAT KO mice. The changes we observed are consistent with cortical ‘hypofrontality’, described in behavioral, pharmacological and neurochemical studies of DAT KO mice. In contrast to the present results, our previous study of the SERT KO mouse revealed a generally more robust cortico-limbic circuit compared to WT mice. These studies indicate that a panel of MR imaging methods combined with statistical parametric mapping analyses is a powerful tool for endophenotyping the pathological manifestations of genetic alterations in executive/reward circuitry.The line of DAT KO mice used in these experiments has been described previously All protocols involving animals in this work were approved by the Institutional Animal Care and Use Committee of the California Institute of Technology.2 with 0.5 mg/ml rhodamine dextran-amine (3k)  was injected unilaterally into the right prefrontal cortex (coordinates x –0.8 mm (from the midline), y +1.9 mm (from Bregma), z 1.8 mm  The stereotaxic injection procedure was similar to that employed by Bearer et al in vivo imaging, animals were sacrificed and brains fixed via transcardiac perfusion with 4% paraformaldehyde (PFA) as previously described Within 10 days after in vivo imaging the animal's head was secured in a Teflon stereotaxic unit within the RF coil to minimize movement and to aid in reproducible placement. Temperature and respiration were continuously monitored during data acquisition and remained within normal ranges. We employed a 3D RARE imaging sequence eff  = 250 ms/12 ms; matrix size of 160×128×78; FOV 16 mm ×12.8 mm ×7.8 mm; yielding 100 µm isotropic voxels with 40 minute scan time.Each animal was scanned before the stereotaxic injection; and beginning at 0∶54±0∶13, 4∶23±0∶09, 8∶13±0∶06, and 25∶45±0∶48 hours post injection. Times are averages over all animals ± standard deviation. We use the midpoint of each 40 minute scan as the “scan time” and for convenience call these the 1 hr, 4 hr, 8 hr, and 26 hr time points. An 11.7 T 89 mm vertical bore Bruker BioSpin Avance DRX500 scanner  equipped with a Micro2.5 gradient system was used to acquire all mouse brain images and spectroscopic data with a 35 mm linear birdcage radio frequency (RF) coil. For in vivo mouse brain magnetic resonance spectroscopy (MRS) experiments were conducted using Point Resolved Spectroscopy (PRESS) 3 volume (8 µl) at the center of the volume used for shimming.All ex vivo imaging, two intact heads were secured in a Teflon® holder and submerged in perfluoropolyether  within a 50 ml vial and imaged. The ambient bore temperature was maintained at 4°C by thermostatically controlled airflow. Diffusion weighted images were acquired using a conventional pulsed-gradient spin echo (PGSE) sequence . An optimized six point icosahedral encoding scheme For After DTI imaging, 5 brains were selected for histological examination, 3 DAT KO and 2 WT littermates. Prior to processing, each brain was released from the skull, post-fixed 1–3 days, and then sent to Neuroscience Associates  for gelatin embedding and frozen sectioning as described 2+ injection MR images were skull-stripped using the Brain Surface Extractor (BSE) within BrainSuite2 http://www.brain-map.org/welcome.do).Pre- and post-MnA deformation field analysis implemented by Thompson and coworkers For each mouse brain the spectrum of the relative amount of metabolites inside the experimental PRESS volume was quantified using the QUEST (quantitation based on quantum estimation) module i; trace, TrD; and fractional anisotropy, FA) were placed into the same space for voxel-wise comparisons using the methods outlined above for in vivo MR images.Reconstruction of the apparent diffusion-weighted images included spatial radial Gaussian filtering (0.25 voxel width) to smooth the co-registration cost-function and improve the SNR of all subsequent calculations. The apparent diffusion tensor was calculated conventionally by inversion of the encoding b-matrix. The b-matrix for each diffusion encoding was determined by numerical simulation of the pulse sequence k-space trajectory in order to account for gradient cross-terms Visualization of the MR images and statistical parametric maps was performed with ResolveRT4  and MRIcro Figure S1Rhodamine-dextran is transported to the amygdala in a wild type animal 16 days after injection in the PFC. The sections was mounted, stained with DAPI for nuclei and imaged for rhodamine and DAPI fluorescence. Alternate sections were Nissl stained. A) Rhodamine-dextran appears in the red channel in neurons of the amygdala and not in other areas in this part of the brain. The DAPI-stained nuclei are shown in blue. B) Nissl-stained section adjacent to the section shown in A) which overlaps the boxed area. These results demonstrate that the location of the injection site was appropriate for the introduction of tracer into the expected forebrain-midbrain pathway. The location of the rhodamine fluorescence is consistent with rhodamine-dextran having arrived via retrograde transport along the axons to the neuronal cell body. Scale bar  = 100 µm.(3.23 MB TIF)Click here for additional data file.Video S1Coronal sections through the statistical parametric maps show the progression of Mn2+ accumulation over time. Gray background is pre-injection MDA, while the colored overlays denote areas with increased intensity (P<0.00025) at 1 hr (green), 4 hr (red), 8 hr (yellow), and 24 hr (blue) compared to the preceding time point. Video shows consecutive sections in the coronal directions for the DAT knockout and wild-type cohorts. Slice locations are shown at the bottom right in millimeters with respect to Bregma. Scale bar  = 1 mm.(7.36 MB MOV)Click here for additional data file.Video S2Sagittal sections through time-series statistical parametric maps. Gray background is pre-injection MDA, while the colored overlays denote areas with increased intensity (P<0.00025) at 1 hr (green), 4 hr (red), 8 hr (yellow), and 24 hr (blue) compared to the preceding time point. Video shows consecutive sections in the sagittal directions for the DAT knockout and wild-type cohorts. Slice locations are shown at the bottom right in millimeters with respect to midline. Scale bar  = 1 mm.(6.34 MB MOV)Click here for additional data file.Video S3Transverse sections through time-series statistical parametric maps. Gray background is pre-injection MDA, while the colored overlays denote areas with increased intensity (P<0.00025) at 1 hr (green), 4 hr (red), 8 hr (yellow), and 24 hr (blue) compared to the preceding time point. Video shows consecutive sections in the transverse directions for the DAT knockout and wild-type cohorts. Slice locations are shown at the bottom right in millimeters with respect the brain surface. Scale bar  = 1 mm.(5.69 MB MOV)Click here for additional data file.Video S4Three dimensional rotations of statistical parametric maps. Three dimensional rotations of statistical parametric maps with Gray background is pre-injection MDA, while the colored overlays denote areas with increased intensity (P<0.00025) at 1 hr (green), 4 hr (red), 8 hr (yellow), and 24 hr (blue) compared to the preceding time point.(9.44 MB MOV)Click here for additional data file.