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Absence of DHTKD1 decreased mitochondrial respiration. (A) Overall oxygen consumption rate (OCR) was significantly lower in KO cells compared to WT (P<0.05) including differences in (B) Basal Respiration (P<0.0001); (C) Maximal Respiration (P=0.0002); (D) Spare Respiratory Capacity (P=0.1912); (E) Proton Leak (P<0.0001); (F) Non-mitochondrial Oxygen Consumption (P<0.0001) and (G) ATP Production (P<0.0001). (H) There were no differences in glycolysis, measured by the extracellular acidification rate (ECAR). Differences assessed by T test ***P < 0.001, ****P < 0.0001. Each assay (n=6 for technical replicates) was repeated for 3 times, and OCR and ECAR values showed in each panel were averaged. ns, Not Statistically Significant.
Source publication
Cardiometabolic disease affects the majority of individuals worldwide. The metabolite α-aminoadipic acid (2-AAA) was identified as a biomarker of Type 2 Diabetes (T2D). However, the mechanisms underlying this association remain unknown. DHTKD1, a central gene in the 2-AAA pathway, has been linked to 2-AAA levels and metabolic phenotypes. However, r...
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Background
Elevated plasma levels of alpha‐aminoadipic acid (2‐AAA) have been associated with the development of type 2 diabetes and atherosclerosis. However, the nature of the association remains unknown.
Methods and Results
We identified genetic determinants of plasma 2‐AAA through meta‐analysis of genome‐wide association study data in 5456 indi...
Citations
... For example, previous studies demonstrated that OGDH can partially compensate for loss of DHTKD1 by metabolizing its substrate [9]. Comparatively, individuals with biallelic variants in either OGDHL or DHTKD1 exhibit a very broad clinical presentation, and neither Dhtkd1 nor Ogdhl knockout mice show severe phenotypes, supporting some degree of functional compensation at play [11,[66][67][68]. Building on this principle, our rescue experiments demonstrate that overexpression of OGDH effectively rescues phenotypes resulting from the loss of Ogdhl or Dhtkd1, while the ability of OGDHL to rescue Ogdh loss is much more limited. ...
Background
Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship.
Methods
Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity.
Results
A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy.
Conclusions
Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as “OGDHL-related disorders”.
... Impaired nucleoside-sugar transporter SLC35A5 is associated with neurological dysfunction [24,25]. Mutations in the mitochondrial regulator DHTK1 is associated with amyotrophic lateral sclerosis [26][27][28][29]. STX3 modulates axonal trafficking and exocytosis [30,31], and we and others reported a link between RBM5 and endocytosis [14,32]. ...
It is not clear if inhibiting the pro-death gene RNA binding motif 5 (RBM5) is neuroprotective in isolated primary neurons or if it regulates cell survival in a sex-dependent manner. Here we established sex-dichotomized primary cortical neuron cultures from transgenic mice harboring a floxed RBM5 gene-trap. Lentivirus-mediated expression of CRE was used to silence RBM5 expression. Male and female neurons were maintained in next-generation Neurobasal-Plus media and subjected to a mechanical stretch-injury (to model traumatic brain injury) or oxygen-glucose deprivation/OGD (to model ischemia). RBM5 KO did not affect 24 h post-injury survival as determined by lactate dehydrogenase (LDH) release, in either paradigm. In contrast, female KO neurons had increased spectrin breakdown products post-insult (in both models). Furthermore, in OGD, RBM5 KO in male neurons exacerbated injury-induced downregulation of pro-survival AKT activation (pAKT473) but conversely led to pAKT473 sparing in female neurons. Moreover, global proteomics identified 19 differentially expressed (DE) proteins in OGD-injured male neurons, and 102 DE proteins in injured female neurons. Two novel RBM5-regulated proteins (PIGQ and EST1C) were identified in injured male KO neurons, and 8 novel proteins identified in injured female KO neurons (S35A5, DHTK1, STX3, IF3M, RN167, K1C14, DYHS, and MED13). In summary, RBM5 inhibition does not modify neuronal survival in primary mouse neurons in 2 clinically relevant models of excitotoxic insult, but RBM5 does regulate intracellular responses to injury in a sex-dependent manner.
... 2-AAA is derived from the breakdown of the essential amino acid lysine, and is primarily metabolized within mitochondria, with potential involvement in oxidative stress (8,9). Elevated 2-AAA is associated with increased insulin secretion, obesity, and dysregulated mitochondrial metabolism (5,7,(10)(11)(12)(13). This makes 2-AAA an interesting novel candidate in cardiometabolic disease biology. ...
... Previous studies have also highlighted an association between 2-AAA and obesity, including both BMI and waist circumference (7,11,41,42). While one study has found that 2-AAA is protective against obesity and diabetes in mice (43), these findings are in contrast to all other studies, and may be related to specific metabolic anomalies in the mouse model used (13,44,1). In our study, 2-AAA associated with increased visceral fat in HATIM, but not subcutaneous or pericardial fat. ...
Introduction
Plasma levels of the metabolite alpha-aminoadipic acid (2-AAA) have been associated with risk of type 2 diabetes (T2D) and atherosclerosis. However, little is known about the relationship of 2-AAA to other cardiometabolic risk markers in pre-disease states, or in the setting of comorbid disease.
Methods
We measured circulating 2-AAA using two methods in 1) a sample of 261 healthy individuals (2-AAA Study), and 2) in a sample of 134 persons comprising 110 individuals with treated HIV, with or without T2D, a population at high risk of metabolic disease and cardiovascular events despite suppression of circulating virus, and 24 individuals with T2D without HIV (HATIM Study). We examined associations between plasma 2-AAA and markers of cardiometabolic health within each cohort.
Results and discussion
We observed differences in 2-AAA by sex and race in both cohorts, with higher levels observed in men compared with women, and in Asian compared with Black or white individuals (P<0.05). There was no significant difference in 2-AAA by HIV status within individuals with T2D in the HATIM Study. We confirmed associations between 2-AAA and dyslipidemia in both cohorts, where high 2-AAA associated with low HDL cholesterol (P<0.001) and high triglycerides (P<0.05). As expected, within the cohort of people with HIV, 2-AAA was higher in the setting of T2D compared to pre-diabetes or normoglycemia (P<0.001). 2-AAA was positively associated with body mass index (BMI) in the 2-AAA Study, and with waist circumference and measures of visceral fat volume in HATIM (all P<0.05). Further, 2-AAA associated with increased liver fat in persons with HIV (P<0.001). Our study confirms 2-AAA as a marker of cardiometabolic risk in both healthy individuals and those at high cardiometabolic risk, reveals relationships with adiposity and hepatic steatosis, and highlights important differences by sex and race. Further studies are warranted to establish molecular mechanisms linking 2-AAA to disease in other high-risk populations.
... The human 2-oxoadipate dehydrogenase (E1a, also known as DHTKD1) is a mitochondrial protein in the L-lysine degradative pathway which is involved in the formation of glutaryl-CoA and NADH (+H + ) from 2-oxoadipate (OA) [1][2][3][4] and is critical for mitochondrial metabolism [5][6][7]. It was recognized that E1a could assemble into a 2-oxoadipate dehydrogenase multienzyme complex (OADHc) by sharing the dihydrolipoamide succinyltransferase (E2o) and dihydrolipoamide dehydrogenase (E3) components with 2-oxoglutarate dehydrogenase (E1o) of the 2-oxoglutarate dehydrogenase complex (OGDHc), suggesting a crosstalk between the OGDHc in the tricarboxylic acid (TCA) cycle and OADHc in L-lysine catabolism [2][3][4]. ...
The human 2-oxoadipate dehydrogenase complex (OADHc) in L-lysine catabolism is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA and NADH (+H+). Genetic findings have linked the DHTKD1 encoding 2-oxoadipate dehydrogenase (E1a), the first component of the OADHc, to pathogenesis of AMOXAD, eosinophilic esophagitis (EoE), and several neurodegenerative diseases. A multipronged approach, including circular dichroism spectroscopy, Fourier Transform Mass Spectrometry, and computational approaches, was applied to provide novel insight into the mechanism and functional versatility of the OADHc. The results demonstrate that E1a oxidizes a non-cognate substrate 2-oxopimelate (OP) as well as OA through the decarboxylation step, but the OADHc was 100-times less effective in reactions producing adipoyl-CoA and NADH from the dihydrolipoamide succinyltransferase (E2o) and dihydrolipoamide dehydrogenase (E3). The results revealed that the E2o is capable of producing succinyl-CoA, glutaryl-CoA, and adipoyl-CoA. The important conclusions are the identification of: (i) the functional promiscuity of E1a and (ii) the ability of the E2o to form acyl-CoA products derived from homologous 2-oxo acids with five, six, and even seven carbon atoms. The findings add to our understanding of both the OADHc function in the L-lysine degradative pathway and of the molecular mechanisms leading to the pathogenesis associated with DHTKD1 variants.
... Hereditary dysfunctions of OADH cause disease states, such as impaired insulin sensitivity (Xu et al., 2018;Xu et al., 2019) or Charcot-Marie-Tooth disease (Xu et al., 2012;Xu et al., 2018;Luan et al., 2020;Castro-Coyotl et al., 2021;Yalcintepe et al., 2021). Large-scale genome association studies reveal that common variants of the human DHTKD1 gene may cause type 2 diabetes and cardiovascular problems (Wu et al., 2014;Plubell et al., 2018;Timmons et al., 2018;Wang et al., 2021). These findings highlight physiological importance of the differences in the 2-oxoadipate saturation of the OADH and OGDH isoenzymes, characterized in the native enzyme complexes enriched from mammalian tissues and in the complexes reconstituted from their components expressed in E.coli (Nemeria et al., 2017;Nemeria et al., 2018). ...
... Specific metabolic action of the OGDHC-and OADHC-directed inhibitors is translated into specific differences in physiological responses to the inhibitors: whereas SP/TESP increases the animal anxiety, the same doses of AP/TEAP increase R-R intervals of ECG ( Figure 3). Regarding the ECG effects of AP/TEAP observed in this work, it is worth noting that a recent study on the knockout of the OADH-encoding DHTKD1, added by the genome-wide association of the common human variants of DHTKD1, found that the gene is linked to cardiovascular risks (Wang et al., 2021). These independent findings are in good accordance with our results on the AP/TMAP effects on ECG (Figures 3, 5). ...
In vitro and in cell cultures, succinyl phosphonate (SP) and adipoyl phosphonate (AP) selectively target dehydrogenases of 2-oxoglutarate (OGDH, encoded by OGDH/OGDHL) and 2-oxoadipate (OADH, encoded by DHTKD1), respectively. To assess the selectivity in animals, the effects of SP, AP, and their membrane-penetrating triethyl esters (TESP and TEAP) on the rat brain metabolism and animal physiology are compared. Opposite effects of the OGDH and OADH inhibitors on activities of OGDH, malate dehydrogenase, glutamine synthetase, and levels of glutamate, lysine, citrulline, and carnosine are shown to result in distinct physiological responses. ECG is changed by AP/TEAP, whereas anxiety is increased by SP/TESP. The potential role of the ester moiety in the uncharged precursors of the 2-oxo acid dehydrogenase inhibitors is estimated. TMAP is shown to be less efficient than TEAP, in agreement with lower lipophilicity of TMAP vs. TEAP. Non-monotonous metabolic and physiological impacts of increasing OADH inhibition are revealed. Compared to the non-treated animals, strong inhibition of OADH decreases levels of tryptophan and beta-aminoisobutyrate and activities of malate dehydrogenase and pyruvate dehydrogenase, increasing the R–R interval of ECG. Thus, both metabolic and physiological actions of the OADH-directed inhibitors AP/TEAP are different from those of the OGDH-directed inhibitors SP/TESP, with the ethyl ester being more efficient than methyl ester.
... The DHTKD1 mutations in humans mostly lack severe phenotypes, but may be associated with muscle weakness and cardiovascular disease risks (3)(4)(5)(6)(7). Some DHTKD1 variants are enriched in patients with eosinophilic esophagitis (8). ...
... A cellular model of the DHTKD1 gene silencing shows disturbed mitochondrial function and biogenesis, associated with decreased activity of the tricarboxylic (TCA) cycle enzyme 2oxoglutarate dehydrogenase (OGDH, EC 1.2.4.2), an isoenzyme of OADH (7,8,20). These data are in accord with the known inactivation of OGDH by 2-oxoadipate in vitro (21). ...
Background
The DHTKD1 -encoded 2-oxoadipate dehydrogenase (OADH) oxidizes 2-oxoadipate—a common intermediate of the lysine and tryptophan catabolism. The mostly low and cell-specific flux through these pathways, and similar activities of OADH and ubiquitously expressed 2-oxoglutarate dehydrogenase (OGDH), agree with often asymptomatic phenotypes of heterozygous mutations in the DHTKD1 gene. Nevertheless, OADH/ DHTKD1 are linked to impaired insulin sensitivity, cardiovascular disease risks, and Charcot-Marie-Tooth neuropathy. We hypothesize that systemic significance of OADH relies on its generation of glutaryl residues for protein glutarylation. Using pharmacological inhibition of OADH and the animal model of spinal cord injury (SCI), we explore this hypothesis.
Methods
The weight-drop model of SCI, a single intranasal administration of an OADH-directed inhibitor trimethyl adipoyl phosphonate (TMAP), and quantification of the associated metabolic changes in the rat brain employ established methods.
Results
The TMAP-induced metabolic changes in the brain of the control, laminectomized (LE) and SCI rats are long-term and (patho)physiology-dependent. Increased glutarylation of the brain proteins, proportional to OADH expression in the control and LE rats, represents a long-term consequence of the OADH inhibition. The proportionality suggests autoglutarylation of OADH, supported by our mass-spectrometric identification of glutarylated K155 and K818 in recombinant human OADH. In SCI rats, TMAP increases glutarylation of the brain proteins more than OADH expression, inducing a strong perturbation in the brain glutathione metabolism. The redox metabolism is not perturbed by TMAP in LE animals, where the inhibition of OADH increases expression of deglutarylase sirtuin 5. The results reveal the glutarylation-imposed control of the brain glutathione metabolism. Glutarylation of the ODP2 subunit of pyruvate dehydrogenase complex at K451 is detected in the rat brain, linking the OADH function to the brain glucose oxidation essential for the redox state. Short-term inhibition of OADH by TMAP administration manifests in increased levels of tryptophan and decreased levels of sirtuins 5 and 3 in the brain.
Conclusion
Pharmacological inhibition of OADH affects acylation system of the brain, causing long-term, (patho)physiology-dependent changes in the expression of OADH and sirtuin 5, protein glutarylation and glutathione metabolism. The identified glutarylation of ODP2 subunit of pyruvate dehydrogenase complex provides a molecular mechanism of the OADH association with diabetes.
Translation of upstream open reading frames (uORFs) typically abrogates translation of main (m)ORFs. The molecular mechanism of uORF regulation in cells is not well understood. Here, we data-mined human and mouse heart ribosome profiling analyses and identified a double-stranded RNA (dsRNA) structure within the GATA4 uORF that cooperates with the start codon to augment uORF translation and inhibits mORF translation. A trans-acting RNA helicase DDX3X inhibits the GATA4 uORF-dsRNA activity and modulates the translational balance of uORF and mORF. Antisense oligonucleotides (ASOs) that disrupt this dsRNA structure promote mORF translation, while ASOs that base-pair immediately downstream (i.e., forming a bimolecular double-stranded region) of either the uORF or mORF start codon enhance uORF or mORF translation, respectively. Human cardiomyocytes and mice treated with a uORF-enhancing ASO showed reduced cardiac GATA4 protein levels and increased resistance to cardiomyocyte hypertrophy. We further show the broad utility of uORF-dsRNA- or mORF-targeting ASO to regulate mORF translation for other mRNAs. This work demonstrates that the uORF-dsRNA element regulates the translation of multiple mRNAs as a generalizable translational control mechanism. Moreover, we develop a valuable strategy to alter protein expression and cellular phenotypes by targeting or generating dsRNA downstream of a uORF or mORF start codon.
Binge drinking is a growing health concern among all age groups. The ability of individuals to handle ethanol in their systems differs not due to differences in enzyme expression but also due to other factors. Vital organs including brain, liver, heart and kidneys are at high risk following repeated exposure to high concentrations of ethanol from binge drinking. The impact of chronic binge drinking on kidneys is not well studied. Using a mouse model of chronic binge drinking, we have identified major metabolic alterations that could set the stage for detrimental effects in the kidneys of male and female mice. We have deciphered that even though there are pathway overlaps, the different sexes exhibited unique and divergent metabolic pathway dysregulations as per the metabolite panels following binge drinking. We have reported that binge drinking could negatively influence renal redox homeostasis in both sexes through regulation of different metabolite clusters. In male mice by downregulation of pantothenic acid and riboflavin synthesis, and female mice by upregulation of α-aminoadipic acid and formyl kynurenine level. Interestingly, uric acid biosynthesis pathway has been upregulated independent of the sex specific effects, portraying the significance of uric acid as a potential marker for binge drinking induced injury in both the sexes. Thus, by altering renal metabolome binge drinking sets the stage for renal damage by disturbing renal redox balance. Our data has high translational potential and will aid in finding ideal therapeutics in a sex-specific manner for subjects exposed to binge drinking induced renal injury.
Understanding the consequences of individual transcriptome variation is fundamental to deciphering human biology and disease. We implement a statistical framework to quantify the contributions of 21 individual traits as drivers of gene expression and alternative splicing variation across 46 human tissues and 781 individuals from the Genotype-Tissue Expression project. We demonstrate that ancestry, sex, age, and BMI make additive and tissue-specific contributions to expression variability, whereas interactions are rare. Variation in splicing is dominated by ancestry and is under genetic control in most tissues, with ribosomal proteins showing a strong enrichment of tissue-shared splicing events. Our analyses reveal a systemic contribution of types 1 and 2 diabetes to tissue transcriptome variation with the strongest signal in the nerve, where histopathology image analysis identifies novel genes related to diabetic neuropathy. Our multi-tissue and multi-trait approach provides an extensive characterization of the main drivers of human transcriptome variation in health and disease.
