2D-GE Image of CHM2A Protein Spot.

<p>Representative 2D-GE images of CHM2A expression over the time course study. Arrows indicate the protein spot of interest. Spots for each genotype and at each time point represent triplicate analysis of a protein pool comprised of n = 4 to 6 cerebella lysates.</p

Summary of 2D-GE Differential Protein Spots.

<p>Venn diagram depicting gel spots that were identified at each time point. Numbers noted in parenthesis are the total number spots with differential intensity identified at that specific time point. Within each time point the breakdown of spots is provided in which the gel intensity suggested either increased (up arrow) or decreased (down arrow) expression relative to the control. A total of 109 spots were differentially expressed. In week one, 22 were unique to this time point whereas 8 were unique to week three and 37 were unique to week five. From the MS analysis, a total of 77 unique proteins were identified in both the mutant and control (paired) gel spots. The protein identifications included 49 increased and 22 decreased.</p

Protein Characterization.

<p>Distribution of differentially expressed proteins by cellular location (A), and biological process (B).</p

CSF-FABP3 concentration in NPC1 patients and controls.

<p>(A) Comparison of FABP3 concentration in untreated (n = 27) and miglustat treated NPC1 patients (n = 18) (p<0.01, t-test, unpaired, Welch's correction). (B) Serial change in FABP3 levels pre- and post-miglustat treatment. Lines connect measurements from the same patient before and after miglustat initiation where one patient was followed serially before miglustat treatment. (C) Percent change in CSF-FABP3 concentration pre- and post-miglustat treatment. (D) Percent change of FABP3 concentration over time in the untreated, treated and pre- post-groups. A one-way ANOVA was used to determined significance (p<0.0001) of the FABP3 concentration change following miglustat initiation.</p

Expression of fatty acid binding protein 3 (FABP3).

<p>(A) Representative 2D-GE images of FABP3 showing the over-expression in the mutant mouse cerebellum compared to the control tissue at the week five time point (top) and graphically displayed (bottom). Arrows note the protein spot of interest. (B) FABP3 levels measured in CSF from control (n = 30) and NPC1 patients (n = 42). Data is represented as average ± standard error of the mean. Significance was determined using an unpaired t-test with Welch's correction, p<0.0001.</p

KEGG Pathway Analysis of Differentially Expressed Proteins.

<p>KEGG pathway analysis of the top ten significant pathways altered in the <i>Npc1^−/−</i> mouse cerebella. The pathway along with the number of proteins associated with the pathway and the p-value are provided.</p

Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

<div><p>A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.</p> </div

Tumor tissue levels of oxidative phosphorylation complex activity and oxidative stress.

<p><b>A</b> Oxidative phosphorylation complex complex activity in SDHB- (n = 4) and VHL-derived (n = 4) PHEO/PGL tissue. * indicates p<0.05. <b>B</b> Malondialdehyde level in SDHB (n = 5) and VHL (n = 5) tissue as a measure of lipid oxidation. <b>C</b> Integrated density of DHE fluorescence in VHL (n = 5), SDHB (n = 6).</p

Oxidative phosphorylation complex activity, reactive oxygen species production, and expression of selected oxidative phosphorylation complex subunits in MPC and MTT. A

<p>Ratio of ADP-stimulated (S3) and baseline oxygen consumption (S4) in MPC and MTT. <b>B</b> Baseline hydrogen peroxide levels (left) and those observed after addition of substrate (glutamate/malate, complex I (center); succinate, complex II (right). ** indicates p<0.01. <b>C</b> protein expression of selected subunits of OXPHOS complexes (complex I: NADH dehydrogenase 1 beta subcomplex subunit 8 (NDUFB8); complex II: succinate dehydrogenase B (SDHB); complex III: ubiquinol-cytochrome-c reductase complex core protein 2 (Uqcrc2); complex V: ATP synthase α (ATPsynα)).</p

Patient Information.

<p>ID (identifier): B followed by a number indicates SDHB, V followed by a number indicates VHL cases. Genetic Background: SDHB^p: SDHB polymorphism, VHL^c: VHL-Chuvash. Gender: F: female, M: male. Type: bp: bilateral primary, mm: metastatic metastases, mltp: multiple primary, pm: primary metastatic, sp: solitary primary. Location: R: right, L: left. Biochem. (biochemical phenotype): A: adrenergic, DA: dopaminergic, NA: noradrenergic, nk: not known. The 4 right columns indicate which samples have been used in the experiments specified by the column headings. Subscript letters are used if more than one tumor from the same patient was available, to indicate which sample has been used (R: right, L: left, A and B: as specified under the heading “location”. OXP (oxidative phosphorylation complex activity), DHE (dihydroethidium fluorescence), MDA (malondialdehyde), WB (western blot). In the western blot column, samples used for LDHA and B and LDHB blots are indicated by . Samples used for all other proteins are indicated by *.</p