Dysregulated placental expression of kynurenine pathway enzymes is associated with inflammation and depression in pregnancy.

BACKGROUND: Perinatal depression (including antenatal-, postnatal-, and depression that spans both timepoints) is a prevalent disorder with high morbidity that affects both mother and child. Even though the full biological blueprints of perinatal depression remain incomplete, multiple studies indicate that, at least for antenatal depression, the disorder has an inflammatory component likely linked to a dysregulation of the enzymatic kynurenine pathway. The production of neuroactive metabolites in this pathway, including quinolinic acid (QUIN), is upregulated in the placenta due to the multiple immunological roles of the metabolites during pregnancy. Since neuroactive metabolites produced by the pathway also may affect mood by directly affecting glutamate neurotransmission, we sought to investigate whether the placental expression of kynurenine pathway enzymes controlling QUIN production was associated with both peripheral inflammation and depressive symptoms during pregnancy. METHODS: 68 placentas obtained at birth were analyzed using qPCR to determine the expression of kynurenine pathway enzymes. Cytokines and metabolites were quantified in plasma using high-sensitivity electroluminescence and ultra-performance liquid chromatography, respectively. Maternal depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale (EPDS) throughout pregnancy and the post-partum. Associations between these factors were assessed using robust linear regression with ranked enzymes. RESULTS: Low placental quinolinate phosphoribosyl transferase (QPRT), the enzyme responsible for degrading QUIN, was associated with higher IL-6 and higher QUIN/kynurenic acid ratios at the 3rd trimester. Moreover, women with severe depressive symptoms in the 3rd trimester had significantly lower placental expression of both QPRT and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD); impaired activity of these two enzymes leads to QUIN accumulation. CONCLUSION: Overall, our data support that a compromised placental environment, featuring low expression of critical kynurenine pathway enzymes is associated with increased levels of plasma cytokines and the dysregulated kynurenine metabolite pattern observed in depressed women during pregnancy.

The effect of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase gene overexpression in the kynurenine pathway on the expression levels of indoleamine 2,3-dioxygenase 1 and interferon-γ in inflammatory conditions: an in vitro study.

BACKGROUND: The kynurenine pathway (KP) can be involved in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. This study aimed to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line to present more information about the effect of this gene on inflammatory conditions and the KP cycle. METHODS AND RESULTS: The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. To simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24 h before transfection, and transfected by Polyethyleneimine (PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and murine Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed 72 h after transfection, and Acmsd expression level increased 29-fold (P < 0.001) in transfected LPS-stimulated cells compared to the control group (LPS-stimulated cells that were not transfected). Additionally, increased Acmsd expression level significantly down-regulated Ifn-γ (P < 0.001) and Ido1 (P < 0.01) expression level in transfected LPS-stimulated cells compared to LPS-stimulated cells. CONCLUSIONS: Acmsd gene overexpression in inflammatory conditions can reduce the expression levels of the Ido1 gene, and its regulator, Ifn-γ. Consequently, it may be considered as a novel regulatory factor in the KP balance.

Quaternary structure of α-amino-ß-carboxymuconate-ϵ-semialdehyde decarboxylase (ACMSD) controls its activity.

α-Amino-ß-carboxymuconate-ϵ-semialdehyde decarboxylase (ACMSD) plays an important role in l-tryptophan degradation via the kynurenine pathway. ACMSD forms a homodimer and is functionally inactive as a monomer because its catalytic assembly requires an arginine residue from a neighboring subunit. However, how the oligomeric state and self-association of ACMSD are controlled in solution remains unexplored. Here, we demonstrate that ACMSD from Pseudomonas fluorescens can self-assemble into homodimer, tetramer, and higher-order structures. Using size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) analysis, we investigated the ACMSD tetramer structure, and fitting the SAXS data with X-ray crystal structures of the monomeric component, we could generate a pseudo-atomic structure of the tetramer. This analysis revealed a tetramer model of ACMSD as a head-on dimer of dimers. We observed that the tetramer is catalytically more active than the dimer and is in equilibrium with the monomer and dimer. Substituting a critical residue of the dimer-dimer interface, His-110, altered the tetramer dissociation profile by increasing the higher-order oligomer portion in solution without changing the X-ray crystal structure. ACMSD self-association was affected by pH, ionic strength, and other electrostatic interactions. Alignment of ACMSD sequences revealed that His-110 is highly conserved in a few bacteria that utilize nitrobenzoic acid as a sole source of carbon and energy, suggesting a dedicated functional role of ACMSD's self-assembly into the tetrameric and higher-order structures. These results indicate that the dynamic oligomerization status potentially regulates ACMSD activity and that SEC-SAXS coupled with X-ray crystallography is a powerful tool for studying protein self-association.

PGC1α regulates ACMSD expression through cooperation with HNF4α.

ACMSD is a tryptophan metabolic key enzyme. HNF4α regulates the transcription of some energy-metabolic enzymes by cooperating with PGC1α, a major transcriptional co-regulator involved in energy metabolism. In this study, we investigated the involvement of PGC1α in Acmsd expression through cooperation with HNF4α. Luciferase reporter assay was performed in NIH3T3 cells using a reporter vector containing HNF4α responsive elements in the Acmsd 5' upstream transcriptional regulatory region together with HNF4α and/or PGC1α expression vectors. The Acmsd luciferase reporter activity was greatly elevated by co-overexpression of HNF4α and PGC1α in NIH3T3 cells. Moreover, the expression level of Acmsd mRNA was significantly increased by co-overexpression of HNF4α and PGC1α in primary hepatocytes compared with expression of either HNF4α or PGC1α alone. These results indicate that PGC1α is involved in Acmsd expression through cooperation with HNF4α.

ACMSD mediated de novo NAD+ biosynthetic impairment in cardiac endothelial cells as a potential therapeutic target for diabetic cardiomyopathy.

OBJECT: The highly conserved α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) is the key enzyme that regulates the de novo NAD+ synthesis from tryptophan. NAD+ metabolism in diabetic cardiomyopathy (DCM) was not elucidated yet. METHODS: Mice were assigned to non-diabetic (NDM) group, streptozocin (STZ)-induced diabetic (DM) group, and nicotinamide (NAM) treated (DM + NAM) group. ACMSD mediated NAD+ metabolism were studied both in mice and patients with diabetes. RESULTS: NAD+ level was significantly lower in the heart of DM mice than that of the NDM group. Supplementation with NAM could partially increased myocardial capillary density and ameliorated myocardial fibrosis by increasing NAD+ level through salvage pathway. Compared with NDM mice, the expression of ACMSD in myocardial endothelial cells of DM mice was significantly increased. It was further confirmed that in endothelial cells, high glucose promoted the expression of ACMSD. Inhibition of ACMSD could increase de novo NAD+ synthesis and improve endothelial cell function by increasing Sirt1 activity. Targeted mass spectrometry analysis indicated increased ACMSD enzyme activity in diabetic patients, higher ACMSD activity increased risk of heart diastolic dysfunction. CONCLUSION: In summary, increased expression of ACMSD lead to impaired de novo NAD+ synthesis in diabetic heart. Inhibition of ACMSD could potentially improve DCM.

Effect of apigenin on tryptophan metabolic key enzymes expression in lipopolysaccharide-induced microglial cells and its mechanism.

[Aims] Flavonoid apigenin (API) has a wide range of biological functions, particularly anti-inflammation. Indoleamine 2,3-dioxygenase (IDO) and 2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) are important tryptophan metabolic enzymes that play pivotal roles in the production of toxic metabolite quinolinic acid. However, the relationship between inflammation and ACMSD remains unclear. The present study investigated the relationship between inflammation and tryptophan metabolic key enzymes. Similarly, the anti-inflammatory effect of API on important tryptophan metabolic enzymes was examined in lipopolysaccharide (LPS)-treated microglial cells. [Main methods] MG6 cells were exposed to LPS with or without API treatment for 24-48 h. IDO and ACMSD mRNA expression and production of inflammatory mediators were analyzed. Activation of inflammatory signaling pathways, such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), was also examined to study the mechanism of API in the inflammatory state. [Key findings] LPS suppressed ACMSD expression and enhanced IDO expression. However, API elevated ACMSD mRNA expression and suppressed IDO mRNA expression in LPS-treated MG6 cells. Furthermore, API suppressed interleukin-6 and nitric oxide production, whereas overproduction of inflammatory mediators enhanced IDO expression and assisted tryptophan degradation. API also inhibited activation of extracellular signal-regulated kinase (Erk) and jun N-terminal kinase (JNK) MAPK, and degradation of IκBα. [Significance] These results indicate alteration of ACMSD expression under inflammatory conditions. Moreover, API recovers expression of tryptophan metabolic key enzymes, which may be mediated by inhibition of proinflammatory mediator production via inactivation of Erk, JNK MAPK, and NF-κB pathways in LPS-stimulated microglial cells.

Structural Basis of Human Dimeric α-Amino-ß-Carboxymuconate-ε-Semialdehyde Decarboxylase Inhibition With TES-1025.

Human α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) stands at a branch point of the de novo NAD+ synthesis pathway and plays an important role in maintaining NAD+ homeostasis. It has been recently identified as a novel therapeutic target for a wide range of diseases, including inflammatory, metabolic disorders, and aging. So far, in absence of potent and selective enzyme inhibitors, only a crystal structure of the complex of human dimeric ACMSD with pseudo-substrate dipicolinic acid has been resolved. In this study, we report the crystal structure of the complex of human dimeric ACMSD with TES-1025, the first nanomolar inhibitor of this target, which shows a binding conformation different from the previously published predicted binding mode obtained by docking experiments. The inhibitor has a K i value of 0.85 ± 0.22 nM and binds in the catalytic site, interacting with the Zn2+ metal ion and with residues belonging to both chains of the dimer. The results provide new structural information about the mechanism of inhibition exerted by a novel class of compounds on the ACMSD enzyme, a novel therapeutic target for liver and kidney diseases.

Shortcutting the diagnostic odyssey: the multidisciplinary Program for Undiagnosed Rare Diseases in adults (UD-PrOZA).

BACKGROUND: In order to facilitate the diagnostic process for adult patients suffering from a rare disease, the Undiagnosed Disease Program (UD-PrOZA) was founded in 2015 at the Ghent University Hospital in Belgium. In this study we report the five-year results of our multidisciplinary approach in rare disease diagnostics. METHODS: Patients referred by a healthcare provider, in which an underlying rare disease is likely, qualify for a UD-PrOZA evaluation. UD-PrOZA uses a multidisciplinary clinical approach combined with state-of-the-art genomic technologies in close collaboration with research facilities to diagnose patients. RESULTS: Between 2015 and 2020, 692 patients (94% adults) were referred of which 329 (48%) were accepted for evaluation. In 18% (60 of 329) of the cases a definite diagnosis was made. 88% (53 of 60) of the established diagnoses had a genetic origin. 65% (39 of 60) of the genetic diagnoses were made through whole exome sequencing (WES). The mean time interval between symptom-onset and diagnosis was 19 years. Key observations included novel genotype-phenotype correlations, new variants in known disease genes and the identification of three new disease genes. In 13% (7 of 53), identifying the molecular cause was associated with therapeutic recommendations and in 88% (53 of 60), gene specific genetic counseling was made possible. Actionable secondary findings were reported in 7% (12 of 177) of the patients in which WES was performed. CONCLUSION: UD-PrOZA offers an innovative interdisciplinary platform to diagnose rare diseases in adults with previously unexplained medical problems and to facilitate translational research.

Modulation of NAD+ biosynthesis activates SIRT1 and resists cisplatin-induced ototoxicity.

Cisplatin, the most widely used platinum-based anticancer drug, often causes progressive and irreversible sensorineural hearing loss in cancer patients. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. Nicotinamide adenine dinucleotide (NAD+), a co-substrate for the sirtuin family and PARPs, has emerged as a potent therapeutic molecular target in various diseases. In our investigates, we observed that NAD+ level was changed in the cochlear explants of mice treated with cisplatin. Supplementation of a specific inhibitor (TES-1025) of α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), a rate-limiting enzyme of NAD+de novo synthesis pathway, promoted SIRT1 activity, increased mtDNA contents and enhanced AMPK expression, thus significantly reducing hair cells loss and deformation. The protection was blocked by EX527, a specific SIRT1 inhibitor. Meanwhile, the use of NMN, a precursor of NAD+ salvage synthesis pathway, had shown beneficial effect on hair cell under cisplatin administration, effectively suppressing PARP1. In vivo experiments confirmed the hair cell protection of NAD+ modulators in cisplatin treated mice and zebrafish. In conclusion, we demonstrated that modulation of NAD+ biosynthesis via the de novo synthesis pathway and the salvage synthesis pathway could both prevent ototoxicity of cisplatin. These results suggested that direct modulation of cellular NAD+ levels could be a promising therapeutic approach for protection of hearing from cisplatin-induced ototoxicity.

Kynurenine pathway in Parkinson's disease-An update.

Parkinson's disease (PD) is a complex multi-factorial neurodegenerative disorder where various altered metabolic pathways contribute to the progression of the disease. Tryptophan (TRP) is a major precursor in kynurenine pathway (KP) and it has been discussed in various in vitro studies that the metabolites quinolinic acid (QUIN) causes neurotoxicity and kynurenic acid (KYNA) acts as neuroprotectant respectively. More studies are also focused on the effects of other KP metabolites and its enzymes as it has an association with ageing and PD pathogenesis. Until now, very few studies have targeted the role of genetic mutations in abnormal KP metabolism in adverse conditions of PD. Therefore, the present review gives an updated research studies on KP in connection with PD. Moreover, the review emphasizes on the urge for the development of biomarkers and also this would be an initiative in generating an alternative therapeutic approach for PD.

ACMSD: A Novel Target for Modulating NAD+ Homeostasis.

NAD+ has a pivotal role in regulating many biological processes. A recent study (Palzer et al., Cell Rep. 2018, 25;1359-1370) demonstrated that alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) is a key regulator of NAD+ metabolism and overexpression of human ACMSD leads to niacin dependency for NAD+ biosynthesis in mice, providing important insights into human diseases associated with niacin/NAD+ deficiency.

Association of three candidate genetic variants in ACMSD/TMEM163, GPNMB and BCKDK /STX1B with sporadic Parkinson's disease in Han Chinese.

Large-scale meta-analyses of genome-wide association studies have identified that polymorphisms ACMSD/TMEM163 rs6430538, GPNMB rs199347 and BCKDK /STX1B rs14235 to be the risk loci for Parkinson's disease (PD) in a Caucasian population. However, the role of these three polymorphisms in a Han Chinese population from mainland China still remains to be clarified. We conducted a large sample study to examine genetic associations of rs6430538, rs199347 and rs14235 with PD in a Han Chinese population of 989 sporadic PD patients and 1058 healthy controls. All subjects were genotyped for these loci using the Sequenom iPLEX Assay. In addition, we conducted further stratified analysis according to age at onset and compared the clinical characteristics between minor allele carriers and non-carriers for each locus. However, no significant differences were found in genotype and allele frequency distribution between PD patients and controls for the three loci, even after being stratified by age at onset. Moreover, we demonstrated that minor allele carriers cannot be distinguished from non-carriers based on their clinical features. Our study is the first to demonstrate that ACMSD/TMEM163 rs6430538, GPNMB rs199347 and BCKDK /STX1B rs14235 do not confer a significant risk for sporadic PD in mainland China. Therefore, more replication studies in additional Chinese population and other cohorts and functional studies are warranted to further clarify the role of the three loci in PD susceptibility.

Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD+ Synthesis.

NAD+ is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD+ synthesis. In contrast, mice easily generate sufficient NAD+ solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD+ deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD+ metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD+ levels in aging and neurodegenerative diseases.

A Novel p.Glu298Lys Mutation in the ACMSD Gene in Sporadic Parkinson's Disease.

BACKGROUND: Common genetic variability in the ACMSD gene has been associated with increased risk for Parkinson's disease (PD) but ACMSD mutations in clinical cases of PD have so far not been reported. OBJECTIVE: To describe a case of sporadic PD carrying a novel ACMSD mutation. METHODS: As part of a genetic study to identify potential pathogenic gene defects related to PD in the Mediterranean island Menorca, an initial group of 62 PD patients underwent mutational screening using a panel-based sequencing approach. RESULTS: We report a 74-years-old man with sporadic PD who developed tremor in his right hand and slowness. On examination, moderate rigidity, asymmetric bradykinesia, and bilateral action tremor were present. He was started on levodopa with significant improvement. Two years later, he developed wearing off phenomena. The genetic study in the patient identified a novel ACMSD mutation resulting in p.Glu298Lys amino-acid change which was not present in neurologically normal population. CONCLUSIONS: Our data suggest that not only common genetic variability but also rare variants in ACMSD alone or in combination with other risk factors might increase the risk of PD.

Is the Enzyme ACMSD a Novel Therapeutic Target in Parkinson's Disease?

Several large genome wide association studies have identified a locus in close proximity to the gene encoding the enzyme aminocarboxymuconate-semialdehyde-decarboxylase (ACMSD) to be associated with the risk for Parkinson's disease (PD), tentatively suggesting that this enzyme might influence PD pathogenesis. Further support for this comes from the recent identification of a disease-segregating stop codon mutation in ACMSD in a family with Parkinsonism, and a missense mutation in the ACMSD gene predicted to disrupt enzyme function in an individual with typical PD. ACMSD is part of the kynurenine pathway, responsible for the catalytic breakdown of tryptophan into NAD+, generating several neuroactive metabolites in the process. The enzyme is located at a key branch-point of the pathway, limiting the production of the neurotoxin quinolinic acid, which has excitotoxic and inflammatory properties. In this review, we discuss the genetic findings in light of the functions of ACMSD and its potential involvement in PD pathogenesis.

Meta-analysis supports association of a functional SNP (rs1801133) in the MTHFR gene with Parkinson's disease.

The MTHFR is a candidate risk gene for Parkinson's disease (PD), and a functional SNP (rs1801133) in the coding region of this gene has been investigated for the associations with the illness extensively among worldwide populations, but overall the results were inconsistent. Here, to assess the relationship between rs1801133 and risk of PD in general populations, we conducted a systematic meta-analysis by combining all available case-control samples in European and Asian populations, with a total of 1820 PD cases and 7530 healthy controls, and the pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for rs1801133 and PD were calculated using the Mantel-Haenszel method with a fixed-effect model. Overall, rs1801133 was significantly associated with the risk of PD (allelic model, pooled OR=1.212 for T allele, 95% CI=1.097-1.340, p-value=0.0002). When stratifying for ethnicity, significant association was also observed in European (allelic model, pooled OR=1.187 for T allele, 95% CI=1.058-1.332, p-value=0.004) and Asian samples (allelic model, pooled OR=1.293 for T allele, 95% CI=1.058-1.580, p-value=0.012) respectively. In addition, rs1801133 was also significantly associated with MTHFR mRNA expression in both CEU (European, p-value=0.0149) and CHB (Chinese, p-value=0.0178) HapMap populations. Collectively, our meta-analysis suggests that rs1801133 is significantly associated with susceptibility to PD in European and Asian populations, and MTHFR is likely an authentic risk gene for PD.