ACO2 deficiency increases vulnerability to Parkinson's disease via dysregulating mitochondrial function and histone acetylation-mediated transcription of autophagy genes.

Parkinson's disease (PD) is characterized by α-synuclein aggregation in dopaminergic (DA) neurons, which are sensitive to oxidative stress. Mitochondria aconitase 2 (ACO2) is an essential enzyme in the tricarboxylic acid cycle that orchestrates mitochondrial and autophagic functions to energy metabolism. Though widely linked to diseases, its relation to PD has not been fully clarified. Here we revealed that the peripheral ACO2 activity was significantly decreased in PD patients and associated with their onset age and disease durations. The knock-in mouse and Drosophila models with the A252T variant displayed aggravated motor deficits and DA neuron degeneration after 6-OHDA and rotenone-induction, and the ACO2 knockdown or blockade cells showed features of mitochondrial and autophagic dysfunction. Moreover, the transcription of autophagy-related genes LC3 and Atg5 was significantly downregulated via inhibited histone acetylation at the H3K9 and H4K5 sites. These data provided multi-dimensional evidences supporting the essential roles of ACO2, and as a potential early biomarker to be used in clinical trials for assessing the effects of antioxidants in PD. Moreover, ameliorating energy metabolism by targeting ACO2 could be considered as a potential therapeutic strategy for PD and other neurodegenerative disorders.

Iron-dependent post transcriptional control of mitochondrial aconitase expression.

Iron regulatory proteins (IRPs) control the translation of animal cell mRNAs encoding proteins with diverse roles. This includes the iron storage protein ferritin and the tricarboxylic cycle (TCA) enzyme mitochondrial aconitase (ACO2) through iron-dependent binding of IRP to the iron responsive element (IRE) in the 5' untranslated region (UTR). To further elucidate the mechanisms allowing IRPs to control translation of 5' IRE-containing mRNA differentially, we focused on Aco2 mRNA, which is weakly controlled versus the ferritins. Rat liver contains two classes of Aco2 mRNAs, with and without an IRE, due to alterations in the transcription start site. Structural analysis showed that the Aco2 IRE adopts the canonical IRE structure but lacks the dynamic internal loop/bulge five base pairs 5' of the CAGUG(U/C) terminal loop in the ferritin IREs. Unlike ferritin mRNAs, the Aco2 IRE lacks an extensive base-paired flanking region. Using a full-length Aco2 mRNA expression construct, iron controlled ACO2 expression in an IRE-dependent and IRE-independent manner, the latter of which was eliminated with the ACO23C3S mutant that cannot bind the FeS cluster. Iron regulation of ACO23C3S encoded by the full-length mRNA was completely IRE-dependent. Replacement of the Aco23C3S 5' UTR with the Fth1 IRE with base-paired flanking sequences substantially improved iron responsiveness, as did fusing of the Fth1 base-paired flanking sequences to the native IRE in the Aco3C3S construct. Our studies further define the mechanisms underlying the IRP-dependent translational regulatory hierarchy and reveal that Aco2 mRNA species lacking the IRE contribute to the expression of this TCA cycle enzyme.

Mitochondrial Aconitase ACO2 Links Iron Homeostasis with Tumorigenicity in Non-Small Cell Lung Cancer.

The ability of a patient tumor to engraft an immunodeficient mouse is the strongest known independent indicator of poor prognosis in early-stage non-small cell lung cancer (NSCLC). Analysis of primary NSCLC proteomes revealed low-level expression of mitochondrial aconitase (ACO2) in the more aggressive, engrafting tumors. Knockdown of ACO2 protein expression transformed immortalized lung epithelial cells, whereas upregulation of ACO2 in transformed NSCLC cells inhibited cell proliferation in vitro and tumor growth in vivo. High level ACO2 increased iron response element binding protein 1 (IRP1) and the intracellular labile iron pool. Impaired cellular proliferation associated with high level ACO2 was reversed by treatment of cells with an iron chelator, whereas increased cell proliferation associated with low level ACO2 was suppressed by treatment of cells with iron. Expression of CDGSH iron-sulfur (FeS) domain-containing protein 1 [CISD1; also known as mitoNEET (mNT)] was modulated by ACO2 expression level and inhibition of mNT by RNA interference or by treatment of cells with pioglitazone also increased iron and cell death. Hence, ACO2 is identified as a regulator of iron homeostasis and mNT is implicated as a target in aggressive NSCLC. IMPLICATIONS: FeS cluster-associated proteins including ACO2, mNT (encoded by CISD1), and IRP1 (encoded by ACO1) are part of an "ACO2-Iron Axis" that regulates iron homeostasis and is a determinant of a particularly aggressive subset of NSCLC.

TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

R2R3-MYB transcription factor FaMYB5 is involved in citric acid metabolism in strawberry fruits.

The citrate content of strawberry fruits affects their organoleptic quality. However, little is known about the transcriptional regulatory mechanisms of citric acid metabolism in strawberry fruits. In this study, the R2R3-MYB transcription factor FaMYB5 was identified and placed in the R2R3-MYB subfamily. FaMYB5 is found in the nucleus and shows tissue- and stage-specific expression levels. Citric acid content was positively correlated with FaMYB5 transcript levels. Upregulated FaMYB5 increased citric acid accumulation in transient FaMYB5-overexpressing strawberry fruits, whereas transient RNA silencing of FaMYB5 in strawberry fruits resulted in a reduction of citric acid content. The role of FaMYB5 was verified using stable transgenic NC89 tobacco. Furthermore, a yeast one-hybrid assay revealed that FaMYB5 influences citric acid accumulation by binding to the FaACO (aconitase), FaGAD (glutamate decarboxylase), and FaCS2 (citrate synthase) promoters. Dual-luciferase assays were used to demonstrate that FaMYB5 could activate FaCS2 expression and repress the transcription levels of FaACO and FaGAD. This study identified important roles of FaMYB5 in the regulation of citric acid metabolism and provided a potential target for improving strawberry fruit taste in horticultural crops.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation.

Iron­sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron­sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5'-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

Multimodal approaches for the improvement of the cellular folding of a recombinant iron regulatory protein in E. coli.

BACKGROUND: During the recombinant protein expression, most heterologous proteins expressed in E. coli cell factories are generated as insoluble and inactive aggregates, which prohibit E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The presence of folding modulators such as exogenous molecular chaperones or osmolytes, as well as process variables such as incubation temperature, inducer concentrations, growth media are all important for cellular folding and are investigated in this study. This study also investigated how the cell's stress response system activates and protects the proteins from aggregation. RESULTS: The cells exposed to osmolytes plus a pre-induction heat shock showed a substantial increase in recombinant aconitase activity when combined with modulation of process conditions. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells. CONCLUSIONS: The recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase production during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

Reactivation of latent tuberculosis through modulation of resuscitation promoting factors by diabetes.

The evidence of an association between diabetes and latent tuberculosis infection (LTBI) remains limited and inconsistent. Thus, the study aims to delineate the role of diabetes in activation of latent tuberculosis infection. Murine model of latent tuberculosis and diabetes was developed, bacillary load and gene expression of resuscitation promoting factors (rpfA-E) along with histopathological changes in the lungs and spleen were studied. Treatment for LTBI [Rifampicin (RIF) + Isoniazid (INH)] was also given to latently infected mice with or without diabetes for 4 weeks. Diabetes was found to activate latent tuberculosis as the colony forming unit (CFU) counts were observed to be > 104 in lungs and spleen. The gene expression of hspX was downregulated and that of rpfB and rpfD was observed to be upregulated in latently infected mice with diabetes compared to those without diabetes. However, no significant reduction in the CFU counts was observed after 4 weeks of treatment with RIF and INH. Diabetes helps in the progression of LTBI to active disease mainly through altered expression of resuscitation promoting factors rpfB and rpfD, which can serve as important targets to reduce the shared burden of tuberculosis and diabetes.

ACO2 clinicobiological dataset with extensive phenotype ontology annotation.

Pathogenic variants of the aconitase 2 gene (ACO2) are responsible for a broad clinical spectrum involving optic nerve degeneration, ranging from isolated optic neuropathy with recessive or dominant inheritance, to complex neurodegenerative syndromes with recessive transmission. We created the first public locus-specific database (LSDB) dedicated to ACO2 within the "Global Variome shared LOVD" using exclusively the Human Phenotype Ontology (HPO), a standard vocabulary for describing phenotypic abnormalities. All the variants and clinical cases listed in the literature were incorporated into the database, from which we produced a dataset. We followed a rational and comprehensive approach based on the HPO thesaurus, demonstrating that ACO2 patients should not be classified separately between isolated and syndromic cases. Our data highlight that certain syndromic patients do not have optic neuropathy and provide support for the classification of the recurrent pathogenic variants c.220C>G and c.336C>G as likely pathogenic. Overall, our data records demonstrate that the clinical spectrum of ACO2 should be considered as a continuum of symptoms and refines the classification of some common variants.

Pleomorphic Xanthoastrocytoma with Oligodendroglioma-Like Areas with Negative 1p19q Co-Deletion

The most common mixed glioma encountered in routine surgical practice is oligoastrocytoma (OA); however, its is currently considered a vanishing entity. The 2016 classification of the World Health Organization (WHO) discourages the diagnosis of tumors as mixed glioma. The recommendations are that diffuse gliomas, including those withmixed or ambiguous histological features, should be subjected tomolecular testing. Dual-genotype OAs are not yet a distinct entity or variant in the classification. We report a case ofmixed glioma: a pleomorphic xanthoastrocytoma (PXA)mixed with an oligodendroglioma. The immunohistochemistry (IHC) pattern of isocitrate dehydrogenase 1 (IDH1) negativity with retained nuclear expression of the alpha-thalassemia x-linked intellectual disability syndrome (ATRX) protein, and 1p19q co-deletion negativity in both the components enabled its identification as a mixed glioma rather than a collision tumor. To the best of our knowledge, the case herein presented is the fourth case of PXA with oligodendroglioma. Out of the other three reported cases, only one was of a collision tumor with a dual genotype, and the other two showed similar molecular signatures in both components. The present article discusses the histological, immunohistochemical and molecular features of the aforementioned case.

Loss of mitochondrial aconitase promotes colorectal cancer progression via SCD1-mediated lipid remodeling.

OBJECTIVE: Mitochondrial aconitase (ACO2) is an essential enzyme that bridges the TCA cycle and lipid metabolism. However, its role in cancer development remains to be elucidated. The metabolic subtype of colorectal cancer (CRC) was recently established. We investigated ACO2's potential role in CRC progression through mediating metabolic alterations. METHODS: We compared the mRNA and protein expression of ACO2 between paired CRC and non-tumor tissues from 353 patients. Correlations between ACO2 levels and clinicopathological features were examined. CRC cell lines with knockdown or overexpression of ACO2 were analyzed for cell proliferation and tumor growth. Metabolomics and stable isotope tracing analyses were used to study the metabolic alterations induced by loss of ACO2. RESULTS: ACO2 decreased in >50% of CRC samples compared with matched non-tumor tissues. Decreased ACO2 levels correlated with advanced disease stage (P < 0.001) and shorter patient survival (P < 0.001). Knockdown of ACO2 in CRC cells promoted cell proliferation and tumor formation, while ectopic expression of ACO2 restrained tumor growth. Specifically, blockade of ACO2 caused a reduction in TCA cycle intermediates and suppression of mitochondrial oxidative phosphorylation, resulting in an increase in glycolysis and elevated citrate flux for fatty acid and lipid synthesis. Increased citrate flux induced upregulation of stearoyl-CoA desaturase (SCD1), which enhanced lipid desaturation in ACO2-deficent cells to favor colorectal cancer growth. Pharmacological inhibition of SCD selectively reduced tumor formation of CRC with ACO2 deficiency. CONCLUSIONS: Our study demonstrated that the rewiring metabolic pathway maintains CRC survival during compromised TCA cycles and characterized the therapeutic vulnerability of lipid desaturation in a meaningful subset of CRC with mitochondrial dysfunction.

Transcriptional Repression of SIRT3 Potentiates Mitochondrial Aconitase Activation to Drive Aggressive Prostate Cancer to the Bone.

Metabolic dysregulation is a known hallmark of cancer progression, yet the oncogenic signals that promote metabolic adaptations to drive metastatic cancer remain unclear. Here, we show that transcriptional repression of mitochondrial deacetylase sirtuin 3 (SIRT3) by androgen receptor (AR) and its coregulator steroid receptor coactivator-2 (SRC-2) enhances mitochondrial aconitase (ACO2) activity to favor aggressive prostate cancer. ACO2 promoted mitochondrial citrate synthesis to facilitate de novo lipogenesis, and genetic ablation of ACO2 reduced total lipid content and severely repressed in vivo prostate cancer progression. A single acetylation mark lysine258 on ACO2 functioned as a regulatory motif, and the acetylation-deficient Lys258Arg mutant was enzymatically inactive and failed to rescue growth of ACO2-deficient cells. Acetylation of ACO2 was reversibly regulated by SIRT3, which was predominantly repressed in many tumors including prostate cancer. Mechanistically, SRC-2-bound AR formed a repressive complex by recruiting histone deacetylase 2 to the SIRT3 promoter, and depletion of SRC-2 enhanced SIRT3 expression and simultaneously reduced acetylated ACO2. In human prostate tumors, ACO2 activity was significantly elevated, and increased expression of SRC-2 with concomitant reduction of SIRT3 was found to be a genetic hallmark enriched in prostate cancer metastatic lesions. In a mouse model of spontaneous bone metastasis, suppression of SRC-2 reactivated SIRT3 expression and was sufficient to abolish prostate cancer colonization in the bone microenvironment, implying this nuclear-mitochondrial regulatory axis is a determining factor for metastatic competence. SIGNIFICANCE: This study highlights the importance of mitochondrial aconitase activity in the development of advanced metastatic prostate cancer and suggests that blocking SRC-2 to enhance SIRT3 expression may be therapeutically valuable. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/1/50/F1.large.jpg.

Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy.

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.

Defects in the rice aconitase-encoding OsACO1 gene alter iron homeostasis.

KEY MESSAGE: Rice aconitase gene OsACO1 is involved in the iron deficiency-signaling pathway for the expression of iron deficiency-inducible genes, either thorough enzyme activity or possible specific RNA binding for post-transcriptional regulation. Iron (Fe) is an essential element for virtually all living organisms. When plants are deficient in Fe, Fe acquisition systems are activated to maintain Fe homeostasis, and this regulation is mainly executed at the gene transcription level. Many molecules responsible for Fe uptake, translocation, and storage in plants have been identified and characterized. However, how plants sense Fe status within cells and then induce a transcriptional response is still unclear. In the present study, we found that knockdown of the OsACO1 gene, which encodes an aconitase in rice, leads to the down-regulation of selected Fe deficiency-inducible genes involved in Fe uptake and translocation in roots, and a decrease in Fe concentration in leaves, even when grown under Fe-sufficient conditions. OsACO1 knockdown plants showed a delayed transcriptional response to Fe deficiency compared to wild-type plants. In contrast, overexpression of OsACO1 resulted in the opposite effects. These results suggest that OsACO1 is situated upstream of the Fe deficiency-signaling pathway. Furthermore, we found that the OsACO1 protein potentially has RNA-binding activity. In vitro screening of RNA interactions with OsACO1 revealed that RNA potentially forms a unique stem-loop structure that interacts with OsACO1 via a conserved GGUGG motif within the loop structure. These results suggest that OsACO1 regulate Fe deficiency response either thorough enzyme activity catalyzing isomerization of citrate, or specific RNA binding for post-transcriptional regulation.

Novel compound heterozygous ACO2 mutations in an infant with progressive encephalopathy: A newly identified neurometabolic syndrome.

BACKGROUND: The ACO2 gene encodes mitochondrial aconitase, the enzyme involved in the second step of the tricarboxylic acid cycle, catalyzing the interconversion of citrate into isocitrate. To date, fewer than 20 families harboring ACO2 mutations have been identified since the first report of a neurodegenerative disorder such as infantile cerebellar retinal degeneration in 2012. Subsequently, various phenotypes, from isolated optic atrophy to spastic paraplegia, have been recognized. Here, we report a case of a newly identified neurometabolic syndrome resulting from novel ACO2 mutations, which expands the genetic spectrum and increases clinical awareness in real-world clinical practice. CASE REPORT: A 2-month-old boy presented with hypotonia, cyanosis, and abnormal eye movements. He had severe psychomotor retardation and intractable seizures manifesting with cyanotic episodes. Diffuse cerebral atrophy and bilateral optic atrophy were noted without cerebellar atrophy. With unremarkable results on comprehensive diagnostic work-up and targeted genetic tests, whole exome sequencing revealed novel compound heterozygous variants in ACO2 (p.Met393Ile and p.Cys448Ser), which were confirmed by Sanger sequencing. Although no definitive signs suggestive of metabolic disturbances or mitochondrial dysfunction have been noted in patients with ACO2 mutations to date, elevated plasma glutamate levels were noted in our case. CONCLUSION: A high index of clinical suspicion and awareness of this disease may aid in the diagnosis of cases with unknown neurodegenerative diseases, facilitated by deep sequencing.

Expanding the clinical and phenotypic heterogeneity associated with biallelic variants in ACO2.

OBJECTIVE: We describe the clinical characteristics and genetic etiology of several new cases within the ACO2-related disease spectrum. Mitochondrial aconitase (ACO2) is a nuclear-encoded tricarboxylic acid cycle enzyme. Homozygous pathogenic missense variants in the ACO2 gene were initially associated with infantile degeneration of the cerebrum, cerebellum, and retina, resulting in profound intellectual and developmental disability and early death. Subsequent studies have identified a range of homozygous and compound heterozygous pathogenic missense, nonsense, frameshift, and splice-site ACO2 variants in patients with a spectrum of clinical manifestations and disease severities. METHODS: We describe a cohort of five novel patients with biallelic pathogenic variants in ACO2. We review the clinical histories of these patients as well as the molecular and functional characterization of the associated ACO2 variants and compare with those described previously in the literature. RESULTS: Two siblings with relatively mild symptoms presented with episodic ataxia, mild developmental delays, severe dysarthria, and behavioral abnormalities including hyperactivity and depressive symptoms with generalized anxiety. One patient presented with the classic form with cerebellar hypoplasia, ataxia, seizures, optic atrophy, and retinitis pigmentosa. Another unrelated patient presented with ataxia but developed severe progressive spastic quadriplegia. Another patient demonstrated a spinal muscular atrophy-like presentation with severe neonatal hypotonia, diminished reflexes, and poor respiratory drive, leading to ventilator dependence until death at the age of 9 months. INTERPRETATION: In this study, we highlight the importance of recognizing milder forms of the disorder, which may escape detection due to atypical disease presentation.

Recessive ACO2 variants as a cause of isolated ophthalmologic phenotypes.

The mitochondrial aconitase gene (ACO2) encodes an enzyme that catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid cycle. Biallelic variants in ACO2 are purported to cause two distinct disorders: infantile cerebellar-retinal degeneration (ICRD) which is characterized by CNS abnormalities, neurodevelopmental phenotypes, optic atrophy and retinal degeneration; and optic atrophy 9 (OPA9), characterized by isolated ophthalmologic phenotypes including optic atrophy and low vision. However, some doubt remains as to whether biallelic ACO2 variants can cause isolated ophthalmologic phenotypes. A review of the literature revealed five individuals from three families who carry biallelic ACO2 variants whose phenotypes are consistent with OPA9. Here, we describe a brother and sister with OPA9 who are compound heterozygous for novel missense variants in ACO2; c.[487G>T];[1894G>A], p.[(Val163Leu)];[(Val632Met)]. A review of pathogenic ACO2 variants revealed that those associated with OPA9 are distinct from those associated with ICRD. Missense variants associated with either OPA9 or ICRD do not cluster in distinct ACO2 domains, making it difficult to predict the severity of a variant based on position alone. We conclude that biallelic variants in ACO2 can cause the milder OPA9 phenotype, and that the OPA9-related ACO2 variants identified to date are distinct from those that cause ICRD.

Mitochondrial aconitase controls adipogenesis through mediation of cellular ATP production.

Mitochondrial aconitase (Aco2) catalyzes the conversion of citrate to isocitrate in the TCA cycle, which produces NADH and FADH2, driving synthesis of ATP through OXPHOS. In this study, to explore the relationship between adipogenesis and mitochondrial energy metabolism, we hypothesize that Aco2 may play a key role in the lipid synthesis. Here, we show that overexpression of Aco2 in 3T3-L1 cells significantly increased lipogenesis and adipogenesis, accompanied by elevated mitochondrial biogenesis and ATP production. However, when ATP is depleted by rotenone, an inhibitor of the respiratory chain, the promotive role of Aco2 in adipogenesis is abolished. In contrast to Aco2 overexpression, deficiency of Aco2 markedly reduced lipogenesis and adipogenesis, along with the decreased mitochondrial biogenesis and ATP production. Supplementation of isocitrate efficiently rescued the inhibitory effect of Aco2 deficiency. Similarly, the restorative effect of isocitrate was abolished in the presence of rotenone. Together, these results show that Aco2 sustains normal adipogenesis through mediating ATP production, revealing a potential mechanistic link between TCA cycle enzyme and lipid synthesis. Our work suggest that regulation of adipose tissue mitochondria function may be a potential way for combating abnormal adipogenesis related diseases such as obesity and lipodystrophy.

Cellular iron sensing and regulation: Nuclear IRP1 extends a classic paradigm.

The classic view is that iron regulatory proteins operate at the post-transcriptional level. Iron Regulatory Protein 1 (IRP1) shifts between an apo-form that binds mRNAs and a holo-form that harbors a [4Fe4S] cluster. The latter form is not considered relevant to iron regulation, but rather thought to act as a non-essential cytosolic aconitase. Recent work in Drosophila, however, shows that holo-IRP1 can also translocate to the nucleus, where it appears to downregulate iron metabolism genes, preparing the cell for a decline in iron uptake. The shifting of IRP1 between states requires a functional mitoNEET pathway that includes a glycogen branching enzyme for the repair or disassembly of IRP1's oxidatively damaged [3Fe4S] cluster. The new findings add to the notion that glucose metabolism is modulated by iron metabolism. Furthermore, we propose that ferritin ferroxidase activity participates in the repair of the IRP1 [3Fe4S] cluster leading to the hypothesis that cytosolic ferritin directly contributes to cellular iron sensing.

Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.