Succinate Dehydrogenase Subunit A Research Articles

SIRT3-Mediated Deacetylation of SDHA Rescues Mitochondrial Bioenergetics Contributing to Neuroprotection in Rotenone-Induced PD Models.

Mitochondrial dysfunction is critically involved in the degeneration of dopamine (DA) neurons in the substantia nigra, a common pathological feature of Parkinson's disease (PD). Previous studies have demonstrated that the NAD+-dependent acetylase Sirtuin 3 (SIRT3) participates in maintaining mitochondrial function and is downregulated in aging-related neurodegenerative disorders. The exact mechanism of action of SIRT3 on mitochondrial bioenergetics in PD pathogenesis, however, has not been fully described. In this study, we investigated the regulatory role of SIRT3-mediated deacetylation of mitochondrial complex II (succinate dehydrogenase) subunit A (SDHA) and its effect on neuronal cell survival in rotenone (ROT)-induced rat and differentiated MN9D cell models. The results revealed that SIRT3 activity was suppressed in both in vivo and in vitro PD models. Accompanying this downregulation of SIRT3 was the hyperacetylation of SDHA, impaired activity of mitochondrial complex II, and decreased ATP production. It was found that the inhibition of SIRT3 activity was attributed to a reduction in the NAD+/NADH ratio caused by ROT-induced inhibition of mitochondrial complex I. Activation of SIRT3 by icariin and honokiol inhibited SDHA hyperacetylation and increased complex II activity, leading to increased ATP production and protection against ROT-induced neuronal damage. Furthermore, overexpression of SDHA also exerted potent protective benefits in cells treated with ROT. In addition, treatment of MN9D cells with the NAD+ precursor nicotinamide mononucleotide increased SIRT3 activity and complex II activity and promoted the survival of cells exposed to ROT. These findings unravel a regulatory SIRT3-SDHA axis, which may be closely related to PD pathology. Bioenergetic rescue through SIRT3 activation-dependent improvement of mitochondrial complex II activity may provide an effective strategy for protection from neurodegeneration.

The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum.

Lentinan alleviates diabetic cardiomyopathy by suppressing CAV1/SDHA-regulated mitochondrial dysfunction

Diabetic cardiomyopathy (DCM), characterized by mitochondrial dysfunction and impaired energetics as contributing factors, significantly contributes to high mortality in patients with diabetes. Targeting key proteins involved in mitochondrial dysfunction might offer new therapeutic possibilities for DCM. Lentinan (LNT), a β-(1,3)-glucan polysaccharide obtained from lentinus edodes, has demonstrated biological activity in modulating metabolic syndrome. In this study, the authors investigate LNT's pharmacological effects on and mechanisms against DCM. The results demonstrate that administering LNT to db/db mice reduces cardiomyocyte apoptosis and mitochondrial dysfunction, thereby preventing DCM. Notably, these effects are fully negated by Caveolin-1 (CAV1) overexpression both in vivo and in vitro. Further studies and bioinformatics analysis uncovered that CAV1 bound with Succinate dehydrogenase subunit A (SDHA), triggering the following ubiquitination and degradation of SDHA, which leads to mitochondrial dysfunction and mitochondria-derived apoptosis under PA condition. Silencing CAV1 leads to reduced apoptosis and improved mitochondrial function, which is blocked by SDHA knockdown. In conclusion, CAV1 directly interacts with SDHA to promote ubiquitination and proteasomal degradation, resulting in mitochondrial dysfunction and mitochondria-derived apoptosis, which was depressed by LNT administration. Therefore, LNT may be a potential pharmacological agent in preventing DCM, and targeting the CAV1/SDHA pathway may be a promising therapeutic approach for DCM.

Deacetylase sirtuin 2 negatively regulates myeloid-derived suppressor cell functions in allograft rejection

Although myeloid-derived suppressor cells (MDSCs) are critical for allograft survival, their regulatory mechanism remains unclear. Herein, our results showed that metabolism sensor sirtuin 2 (SIRT2) negatively regulates the functions of MDSCs in inducing allogeneic skin graft rejection. Genetic deletion of SIRT2 in myeloid cells (Sirt2Δmye) increased the number of CD11b+Gr1+ MDSCs in bone marrow, spleens, draining lymph nodes, and allografts, inhibited the production of proinflammatory cytokine tumor necrosis factor ɑ, enhanced the production of anti-inflammatory cytokine interleukin 10, and potentiated the suppressive activation of MDSCs in prolonging allograft skin survival. C-X-C motif chemokine receptor 2 is critical for mediating the recruitment and cytokine production of MDSCs induced by SIRT2. Mechanistically, Sirt2Δmye enhanced NAD+ levels, succinate dehydrogenase subunit A (SDHA) activities, and oxidative phosphorylation (OXPHOS) levels in MDSCs after transplantation. Pharmacologically blocking nicotinamide phosphoribosyltransferase effectively reverses the production of cytokines and suppressive activities of MDSC induced by Sirt2Δmye. Blocking OXPHOS with knockdown of SDHA or pharmacological blocking of SDHA significantly restores Sirt2Δmye-mediated stronger MDSC suppressive activity and inflammatory factor productions. Thus, our findings identify a previously unrecognized interplay between NAD+ and SDH-mediated OXPHOS metabolic pathways in regulating MDSC functions induced by the metabolic sensor SIRT2 in allogeneic transplantation.

Perinatal exposure to high concentration glyphosate-based herbicides induces intestinal apoptosis by activating endoplasmic reticulum stress in offspring

Glyphosate-based herbicides (GBHs), the most widely used pesticide worldwide, have been reported to impair organ function in humans and animals. However, research on the effect of maternal GBHs exposure on the intestinal health of offspring has received little attention. Based on the glyphosate limits defined by Codex Alimentarius Commission and European Food Safety Authority, this study established pregnant sow exposure models to investigate the influence of low (L-GBHs, 20 mg/kg) and high concentration GBHs (H-GBHs, 100 mg/kg) on the intestinal health of offspring and proposed the protective mechanism mediated by betaine. The results showed that the intestinal morphology and barrier function of suckling piglets were damaged in the H-GBHs group. H-GBHs increased the activity of glutathione peroxidase (GPX) and levels of methane dicarboxylic aldehyde (MDA), hydrogen peroxide (H2O2) and inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10)) in suckling piglets and activated Nrf2-mediated antioxidant signaling pathway. Subsequently, we found that exposure to H-GBHs triggered endoplasmic reticulum stress (ERS) and further induced apoptosis by upregulating the expression of Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and Caspase12. Moreover, H-GBHs exposure perturbed mitochondrial membrane fusion and electron transport in mitochondrial respiratory chains by increasing the mRNA expression of mitofusin-2 (MFN2) and succinate dehydrogenase subunit A (SDHA), causing mitochondrial dysfunction. Dietary supplementation with betaine provided modest protection against GBHs-induced intestinal damage in suckling piglets. These findings reveal the mechanism of GBHs-induced intestinal damage in offspring, improving our understanding of the risk of GBHs exposure in pregnant women and suggesting the potential protective effects of betaine against GBHs poisoning.

ODP042 Hereditary Pheochromocytoma with a Novel Mutation in the Succinate Dehydrogenase Subunit A gene

Abstract Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumorswith a diverse clinical presentation. Pathogenic variants in the genes encoding different subunits of the succinate dehydrogenaseenzyme complex that plays a central role in energy metabolism,have been linked to hereditary PPGL syndromes. Here wereport a rare case of hereditary pheochromocytoma with a novel mutation in the succinate dehydrogenase subunit A (SDHA) gene. A middle aged woman presented with left sided abdominal pain and was incidentally found to have bilateral adrenal lesions on abdominal imaging. She denied any symptoms of catecholamine excess but her plasma metanephrines level was elevated. Family history was significant for breast cancer in her motherand sister. Computed tomography (CT) scan of the abdomen showed bilateralbulky adrenal glands with a lesion in theleft adrenal gland. Magnetic resonance imaging (MRI) abdomen was performed for further characterization, the lesion measured approximately 2.8×2.6 cm with imaging characteristics suggestive of pheochromocytoma. Iodine-131 Metaiodobenzylguanidine (MIBG) whole body scan showed abnormal focal radiotracer uptake at the left adrenal gland, and she then underwentleft sided robotic adrenalectomy. Following surgery, the patient had symptomatic relief. Histopathology confirmed the diagnosis of pheochromocytoma measuring 2×2×1.5 cm with PASS (Pheochromocytoma of the Adrenal gland Scaled Score) score of 1/10, due to cellular monotony. Genetic testing revealed that she was positive for a pathogenic mutation in the SDHA gene consistent with the diagnosis of hereditary PPGL syndrome. Genetic testing was performed for the patient due to her presentation with bilateral adrenal masses and family history of cancers. Guidelines recommend genetic testing for patients with features that indicate a high likelihood of a hereditary cause for PPGL, including positive family history, syndromic features, and multifocal, bilateral, or metastatic disease. SDHA mutated PPGLs have diverse phenotypes and may be at high risk of metastasis. They are also associated with extra-adrenal tumors, include gastrointestinal stromal tumours (GIST), pituitary adenomas, papillary thyroid carcinoma, renal carcinoma and breast carcinoma. The detection of susceptibility genes for hereditary PPGL syndromes has key implications, for surveillance to detectextra-adrenal disease and recurrent tumors, as well as for consideration of genetic testing forfamily members. Presentation: No date and time listed

Exploring the effects of artesunate and fuzheng huayu decoction on mitochondria in the treatment of schistosomiasis liver fibrosis

Objective: To compare the effects of artesunate (Art) and fuzheng huayu decoction on mitochondrial autophagy in the treatment of schistosomiasis liver fibrosis. Methods: Eighty C57BL/6 female mice were randomly divided into healthy control group, infection group, Art treatment group and Fuzheng Huayu Decoction treatment group, with 20 mice in each group. Mice in the infection group and treatment group were infected with 16 Schistosoma japonicum cercariae. After 6 weeks, praziquantel (300 mg/kg) was used for 2 days to kill the worms. The Art treatment group was treated with intraperitoneal injection of 100 mg/kg/day, while the Fuzheng Huayu Decoction treatment group was fed 16g of fuzheng huayu decoction per 1kg per day. After 6 weeks, fresh liver tissues of the four groups were collected. Masson staining and Western blot were used to observe the succinate dehydrogenase subunit A (SDHA) and malate dehydrogenase (MDH2), citrate synthase (CS), ketoglutarate dehydrogenase (OGDH), and target of rapamycin 1 (mTORC1) pathway involved in mitochondrial tricarboxylic acid cycle in liver tissues. The relative expression levels of adenylate activated protein kinase (AMPK) and mitochondrial autophagy pathway kinase (PINK1) were detected. Liver tissue samples were extracted from each group to detect the mitochondrial oxygen consumption rate. Two-way ANOVA was used to compare the significance and difference between two sets of samples. Results: Masson staining showed that the infection group mice had significantly higher liver fibrosis area than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group mice had lower liver fibrosis area than the infection group. Western blot analysis showed that the infection group (0.82 ± 0.05) had significantly lower relative expression of SDHA protein than the healthy control group (1.00 ± 0.05) (t = 11.23, P = 0.0035), while the Art treatment group (0.73 ± 0.05) had significantly higher relative expression of SDHA protein than the infection group (t = 10.79, P = 0.0073). However, there was no significant change in Fuzheng Huayu Decoction treatment group (0.98±0.05) (t = 1.925, P = 0.1266). The relative expression of p-AMPK protein was significantly higher in the infection group (1.15 ±0.05) than in the healthy control group (0.98 ± 0.07, t = 12.18, P = 0.0029), and the expression of p-AMPK in the Art treatment group (0.50 ± 0.05) was significantly lower than the infection group (t = 11.78, P = 0.0032). The relative protein expression of AMPK was significantly lower in the infection group (0.80 ± 0.05) than in the healthy control group (1.00 ± 0.05, t = 10.53, P = 0.0046). The expression of AMPK was significantly lower in the Art treatment group (0.54 ± 0.05) than in the infection group (T = 13.98, P = 0.0036). The relative expression of p-mTORC1 protein (0.93 ± 0.08) was not significantly different in the infection group than in the healthy control group (t = 2.28, P = 0.065), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1 protein than the infection group (t = 10.58, P = 0.029). The expression of p-mTORC1/ m-TORC1 was not significantly different in the infection group (0.98 ± 0.03) than in the healthy control group (0.97 ± 0.03, t = 0.98, P = 0.085), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1/ m-TORC1 than the infection group (t = 14.58, P = 0. 009). The relative protein expression of PINK1 was significantly lower in the infection group (0.55 ± 0.05) than in the healthy control group (1.00 ± 0.03, t = 13.49, P = 0.0011), while the Art treatment group (1.21 ± 0.05, t = 9.98, P = 0.0046) and Fuzheng Huayu Decoction treatment group (1.31 ±0.35, t = 6.98, P = 0.027) had significantly higher relative protein expression of PINK1 than the infection group. Mitochondrial function tests showed that after adding substrate complex II, the oxygen consumption of the infection group was lower than the healthy control group, while the Art treatment group and the Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. The oxygen consumption was significantly lower after adding the substrate complex III in the infection group than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. Conclusion: Art can alleviate schistosomiasis liver fibrosis by inhibiting AMPK/mTORC1 signaling pathway activity and enhancing mitochondrial oxygen consumption, autophagy and SDHA expression.

Pharmaceutical targeting of succinate dehydrogenase in fibroblasts controls bleomycin-induced lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix in the lung with fibroblast-to-myofibroblast transition, leading to chronically compromising lung function and death. However, very little is known about the metabolic alterations of fibroblasts in IPF, and there is still a lack of pharmaceutical agents to target the metabolic dysregulation. Here we show a glycolysis upregulation and fatty acid oxidation (FAO) downregulation in fibroblasts from fibrotic lung, and perturbation of glycolysis and FAO affects fibroblasts transdifferentiation. In addition, there is a significant accumulation of succinate both in fibrotic lung tissues and myofibroblasts, where succinate dehydrogenase (SDH) operates in reverse by reducing fumarate to succinate. Then succinate contributes to glycolysis upregulation and FAO downregulation by stabilizing HIF-1α, which promotes the development of lung fibrosis. In addition, we identify a near-infrared small molecule dye, IR-780, as a targeting agent which stimulates mild inhibition of succinate dehydrogenase subunit A (SDHA) in fibroblasts, and which inhibits TGF-β1 induced SDH and succinate elevation, then to prevent fibrosis formation and respiratory dysfunction. Further, enhanced cell retention of IR-780 is shown to promote severe inhibition of SDHA in myofibroblasts, which may contribute to excessive ROS generation and selectively induces myofibroblasts to apoptosis, and then therapeutically improves established lung fibrosis in vivo. These findings indicate that targeting metabolic dysregulation has significant implications for therapies aimed at lung fibrosis and succinate dehydrogenase is an exciting new therapeutic target to treat IPF.

High Expression of Succinate Dehydrogenase Subunit A Which Is Regulated by Histone Acetylation, Acts as a Good Prognostic Factor of Multiple Myeloma Patients.

Most patients with multiple myeloma (MM) will eventually relapse and current treatments have limited effect. Herein, we demonstrate that succinate dehydrogenase subunit A (SDHA) was low expressed in MM patients, and patients with SDHA relatively high expression had long overall survival and progression-free survival. Furthermore, SDHA high expression inhibited proliferation and invasion in MM cell lines and enhanced the anti-tumor and synergistic effect of chemotherapeutics. More importantly, chidamide was proved effective in MM by targeting SDHA, and expression of SDHA was increased by chidamide through acetylating H3K27 site of SDHA. Collectively, high expression of SDHA, which was regulated by histone acetylation and targeted by chidamide, might become a good prognostic factor of MM patients.

SDHA-mediated Warburg effect in malignantly transformed human bronchial epithelial cells following long-term exposure to radon.

Radon and its progeny have been classified as human class I carcinogens by the IARC. However, the mechanisms by which radon induces lung and other cancers, especially the radon-induced Warburg effect, have not been fully elucidated. The aim of this study was to investigate the role of the succinate dehydrogenase subunit A (SDHA)-mediated Warburg effect in (human bronchial epithelial) BEAS-2B cells with malignant transformations induced by long-term radon exposure. Soft agar colony formation and MMP-9 were increased following radon-induced malignant transformation. Additionally, we observed the Warburg effect in BEAS-2B cells following long-term radon exposure, evidenced by increases in the levels of glucose uptake, lactate, and lactate dehydrogenase (LDH). Following radon exposure, the expression of SDHA was decreased, while the levels of HIF-1α and hexokinase-2 (HK2) were increased. Our findings suggested that the SDHA-associated pathway may be involved in mediating the Warburg effect in radon-induced malignant transformation of BEAS-2B.

Molecular Alterations in Dog Pheochromocytomas and Paragangliomas.

Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS.

Abstract 3553: Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer (comprising 80-85 %), is a leading cause of cancer-related death worldwide. KRAS mutation is s major driver oncogene in NSCLC. Constitutive activation of K-Ras via oncogenic mutations plays a key role in development and progression of NSCLC, and is an important target for drug development. Indeed, drugs that target RAF-MAPK and PI3K, the well-established downstream pathways of K-Ras, are being evaluated in the clinic. However, to date, no effective treatments specifically targeting K-Ras have reached the clinic. A new direction for anti-Ras therapies targeting Ras-mediated altered cellular metabolism is implied by recent studies. Hence, as an attempt to investigate novel therapeutic targets to suppress KRAS-driven NSCLC, we assessed metabolic changes in NSCLC cell line and mouse model systems with oncogenic K-Ras mutations. We show that activation of Ras causes metabolic alterations, leading to aerobic glycolysis. Interestingly, despite the changes in the glycolytic pathway, NSCLC cell line and mouse models carrying K-Ras mutation revealed an elevated mitochondrial membrane potential, superoxide production as well as maximal oxygen consumption rate. Moreover, blockade of mitochondrial respiration effectively reduced mutant KRas-driven neoplastic phenotypes in NSCLC cell lines vitro and lung tumor formation in vivo. In contrast, blockade of glycolysis was not effective in the mutant KRas-driven tumorigenic activities. Mechanistically, mutant KRas-mediated activation of Akt2 led to increased expression of succinate dehydrogenase subunit A (SDHA) through increased protein stability by blocking proteasome-mediated degradation, resulting in mitochondrial complex II-dependent respiration. Our study suggests that induction of mitochondrial complex II-dependent respiration is an important mechanism by which oncogenic mutant KRas induces metabolic changes and ROS stress in NSCLC cells, contributing to lung carcinogenesis. Our findings elucidate potential metabolic targets that may provide novel therapeutic strategy for treating NSCLC with oncogenic KRAS mutations. Citation Format: Kwan-Hee Park, Ho-Young Lee. Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3553. doi:10.1158/1538-7445.AM2017-3553

Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.

Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae.

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring mRNA expression. To facilitate gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to stable housekeeping genes is mandatory. In this study, ten housekeeping genes, including beta-actin (Actin) , elongation factor 1 α (EF1A) , glyceralde hyde-3-phosphate dehydrogenase (GAPDH) , ribosomal protein L13 (RPL13) , ribosomal protein 49 (RP49) , α-tubulin (Tubulin) , vacuolar-type H+-ATPase (v-ATPase) , succinate dehydrogenase subunit A (SDHA) , 28S ribosomal RNA (28S) , and 18S ribosomal RNA (18S) from the two-spotted spider mite, Tetranychus urticae, were selected as the candidate reference genes. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the performance of these candidates as endogenous controls across different developmental stages. In addition, RefFinder, which integrates the above-mentioned software tools, provided the overall ranking of the stability/suitability of these candidate reference genes. Among them, PRL13 and v-ATPase were the two most stable housekeeping genes across different developmental stages. This work is the first step toward establishing a standardized qRT-PCR analysis in T. urticae following the MIQE guideline. With the recent release of the T. urticae genome, results from this study provide a critical piece for the subsequent genomics and functional genomics research in this emerging model system.

Genetics of hereditary head and neck paragangliomas

The purpose of this study was to give an overview on hereditary syndromes associated with head and neck paragangliomas (HNPGs). Our methods were the review and discussion of the pertinent literature. About one third of all patients with HNPGs are carriers of germline mutations. Hereditary HNPGs have been described in association with mutations of 10 different genes. Mutations of the genes succinate dehydrogenase subunit D (SDHD), succinate dehydrogenase complex assembly factor 2 gene (SDHAF2), succinate dehydrogenase subunit C (SDHC), and succinate dehydrogenase subunit B (SDHB) are the cause of paraganglioma syndromes (PGLs) 1, 2, 3, and 4. Succinate dehydrogenase subunit A (SDHA), von Hippel-Lindau (VHL), and transmembrane protein 127 (TMEM127) gene mutations also harbor the risk for HNPG development. HNPGs in patients with rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and MYC-associated factor X (MAX) gene mutations have been described very infrequently. All patients with HNPGs should be offered a molecular genetic screening. This screening may usually be restricted to mutations of the genes SDHD, SDHB, and SDHC. Certain clinical parameters can help to set up the order in which those genes should be tested.

Means, Motive, and Opportunity: SDH Mutations Are Suspects in Pituitary Tumors

These are heady times in endocrinology, with the application of new techniques revolutionizing our understanding of the genetic pathophysiology of many syndromes of endocrine deficiency and excess. It seems that not a month goes by without a new genetic discovery, and last year’s teaching slides are already looking out of date. This ferocious pace of progress is uncovering pathways and potential targets for treatment in many rare conditions. Some discoveries are entirely new genetic causes, whereas others are known genes in new disease settings. This situation is analogous to the work of criminologists using the selfsame genetic techniques to link unsolved “cold” cases to known culprits via studies of archived evidence. In the medical setting, similar good clinical detective work is leading to known genetic culprits in other neuroendocrine tumors being hauled in for interrogation in connection with their links to pituitary tumors. In this issue of the JCEM, Dwight et al (1) describe the occurrence of a pituitary adenoma in the setting of a familial succinate dehydrogenase subunit A (SDHA) gene mutation associated with paraganglioma in a first-degree relative. This finding expands the field of the tumor features associated with succinate dehydrogenase subunit (SDHx) mutations into the pituitary, joining the first report from the Stratakis group (2) that definitively linked a succinate dehydrogenase subunit D (SDHD) germline mutation to acromegaly. Also, a case from Spain associated with a succinate dehydrogenase subunit C (SDHC) germline mutation was reported, although without pituitary tumoral DNA confirmation (3). Data from the literature back to 1952 that were reviewed by Xekouki and Stratakis (4) have included 29 instances of coexisting pituitary adenomas and pheochromocytomas/paragangliomas. Some modern cases that were negative for existing causative gene mutations appear to be persuasive for further investigation of SDHx status (4). Indeed, the clinical impact of these mitochondrial protein abnormalities already encompasses a remarkably wide range of tumors from renal cell cancers to sporadic and syndromic gastrointestinal stromal tumors (Carney triad and Carney-Stratakis syndrome) and beyond (5–9). The addition of pituitary adenomas to the expanding phenotype related to inheritable pheochromocytoma/paraganglioma syndromes is intriguing and represents both an opportunity and a challenge. An important area for future study will be to define whether SDHx mutation-associated pituitary adenomas have particular features that differentiate them from the general sporadic population. Clinically relevant pituitary adenomas occur is about 1 in 1000 people in the general population, and prolactinomas account for up to twothirds of these (10). Pituitary adenomas that occur in recognized genetic or familial settings account for about 5% of cases in our experience (11). Most occur in the setting of familial isolated pituitary adenoma—either with or without a germline aryl hydrocarbon receptor interacting protein (AIP) gene mutation—and multiple endocrine neoplasia (MEN) 1 (11–13). Rarer syndromes like that of Carney complex due to protein kinase A regulatory subunit 1A (PRKAR1A) mutations or MEN4 due to cyclindependent kinase inhibitor 1B (CDKN1B) gene mutations also feature pituitary adenomas as part of the described clinical spectrum (14, 15). Large collaborative studies of pituitary adenomas that occur against these known genetic backgrounds have revealed features related to more aggressive behavior, which are useful pointers when determining which patients to offer genetic testing to. AIP

FamilialSDHAMutation Associated With Pituitary Adenoma and Pheochromocytoma/Paraganglioma

Reports of the coexistence of pituitary adenomas and pheochromocytoma/paraganglioma are uncommon. Recently germline mutations in 2 of the genes encoding succinate dehydrogenase, SDHC and SDHD, were associated with pituitary tumors. Our aim was to determine whether the development of a pituitary adenoma was associated with SDHA mutation. A 46-year-old female presented with carotid body paraganglioma (proband). Subsequently the proband's son was diagnosed with a nonfunctioning pituitary macroadenoma at age 30 years. An immunohistochemical analysis of the resected paraganglioma and pituitary adenoma revealed the loss of succinate dehydrogenase subunit B and succinate dehydrogenase subunit A (SDHA) expression in both tumors, with the preservation of staining in nonneoplastic tissue. Mutation analysis showed a novel SDHA mutation (c.1873C>T, p.His625Tyr) in the germline of the proband as well as in the proband's son. In the paraganglioma of the proband, in addition to the germline mutation, a somatic mutation was observed (c.1865G>A, p.Trp622*). In the pituitary adenoma of the proband's son, loss of SDHA immunoreactivity was paradoxically accompanied by loss of the mutant allele. This is the first report of a pituitary adenoma arising in the setting of germline SDHA mutation. The loss of SDHA protein expression in both the paraganglioma (proband) and pituitary adenoma (proband's son) argues strongly for a causative role of SDHA mutation. This report further strengthens the link between pituitary neoplasia and germline SDH mutation. Although pituitary adenomas appear rare among patients carrying SDH subunit mutations, they may have been underrecognized due to the low penetrance of disease and lack of systematic surveillance.

Immunohistochemical Loss of Succinate Dehydrogenase Subunit A (SDHA) in Gastrointestinal Stromal Tumors (GISTs) Signals SDHA Germline Mutation

A subset (7% to 10%) of gastric gastrointestinal stromal tumors (GISTs) is notable for the immunohistochemical loss of succinate dehydrogenase (SDH) subunit B (SDHB), which signals the loss of function of the SDH complex consisting of mitochondrial inner membrane proteins. These SDH-deficient GISTs are known to be KIT/PDGFRA wild type, and most patients affected by this subset of GISTs are young. Some of these patients have germline mutations of SDH subunit genes SDHB, SDHC, or SDHD, known as Carney-Stratakis syndrome when combined with paraganglioma. More recently, germline mutations in SDH subunit A gene (SDHA) have also been reported in few patients with KIT/PDGFRA wild-type GISTs. In this study we immunohistochemically examined 127 SDHB-negative and 556 SDHB-positive gastric GISTs and 261 SDHB-positive intestinal GISTs for SDHA expression using a mouse monoclonal antibody 2E3 (Abcam). Cases with available DNA were tested for SDHA, SDHB, SDHC, and SDHD gene mutations using a hybridization-based custom capture next-generation sequencing assay. A total of 36 SDHA-negative GISTs (28%) were found among 127 SDHB-negative gastric GISTs. No SDHB-positive GIST was SDHA negative. Among 7 SDHA-negative tumors analyzed, there were 7 SDHA mutants, most germline. A second hit indicating biallelic inactivation of SDHA was present in 6 of those cases. These patients had no other SDH subunit gene mutations. Among the 25 SDHA-positive, SDHB-negative GISTs analyzed, we identified 3 SDHA mutations (1 germline), and 11 SDHB, SDHC, or SDHD mutations (mostly germline), and 11 patients with no SDH mutations. Compared with patients with SDHA-positive GISTs, those with SDHA-negative GISTs had an older median age (34 vs. 21 y), lower female to male ratio (1.8 vs. 3.1) but similar mitotic counts and median tumor sizes, with a slow course of disease in most cases, despite a slightly higher rate of liver metastases. SDHA-negative GISTs comprise approximately 30% of SDHB-negative/SDH-deficient GISTs, and SDHA loss generally correlates with SDHA mutations.