10-formyltetrahydrofolate Dehydrogenase Research Articles

Accumulation of Cerebrospinal Fluid, Ventricular Enlargement, and Cerebral Folate Metabolic Errors Unify a Diverse Group of Neuropsychiatric Conditions Affecting Adult Neocortical Functions.

Cerebrospinal fluid (CSF) is a fluid critical to brain development, function, and health. It is actively secreted by the choroid plexus, and it emanates from brain tissue due to osmolar exchange and the constant contribution of brain metabolism and astroglial fluid output to interstitial fluid into the ventricles of the brain. CSF acts as a growth medium for the developing cerebral cortex and a source of nutrients and signalling throughout life. Together with perivascular glymphatic and interstitial fluid movement through the brain and into CSF, it also acts to remove toxins and maintain metabolic balance. In this study, we focused on cerebral folate status, measuring CSF concentrations of folate receptor alpha (FOLR1); aldehyde dehydrogenase 1L1, also known as 10-formyl tetrahydrofolate dehydrogenase (ALDH1L1 and FDH); and total folate. These demonstrate the transport of folate from blood across the blood-CSF barrier and into CSF (FOLR1 + folate), and the transport of folate through the primary FDH pathway from CSF into brain FDH + ve astrocytes. Based on our hypothesis that CSF flow, drainage issues, or osmotic forces, resulting in fluid accumulation, would have an associated cerebral folate imbalance, we investigated folate status in CSF from neurological conditions that have a severity association with enlarged ventricles. We found that all the conditions we examined had a folate imbalance, but these folate imbalances were not all the same. Given that folate is essential for key cellular processes, including DNA/RNA synthesis, methylation, nitric oxide, and neurotransmitter synthesis, we conclude that ageing or some form of trauma in life can lead to CSF accumulation and ventricular enlargement and result in a specific folate imbalance/deficiency associated with the specific neurological condition. We believe that addressing cerebral folate imbalance may therefore alleviate many of the underlying deficits and symptoms in these conditions.

Abstract LB_B03: Lentivirus-induced ALDH1L1 knock-in inhibits proliferation and migration and activates apoptosis of human A549 lung adenocarcinoma cells

Abstract Lung Adenocarcinoma (LUAD), one of the most common life-threatening malignancies worldwide, represents about 40% of all lung cancers. Human Protein Atlas and OncoLnc database indicate that LUAD tumors have significantly lower mRNA levels of ALDH1L1 (10-formyltetrahydrofolate dehydrogenase, a regulatory enzyme in folate metabolism) than in non-tumor lung tissues. Moreover, higher intratumoral ALDH1L1 expression in LUAD patients correlates with better clinical outcome. These findings raise the question of whether ALDH1L1 knock-in can have a therapeutic effect. As the first step in addressing this question, we knocked-in ALDH1L1 in A549 lung adenocarcinoma cells using the lentiviral delivery. Of note, as most cancer cell lines, A549 cells do not express ALDH1L1. We have constructed the lentiviral vector carrying ALDH1L1 and green fluorescent protein (EGFP) cDNA under the control of CMV and SV40 promoters, respectively. Recombinant lentivirus (Lv-ALDH1L1), generated and packed in HEK293 cells, was used to transduce A549 cells. The efficiency of the transduction was evaluated by monitoring the GFP expression using the fluorescence microscopy of live cells. The expression of ALDH1L1 was determined at the mRNA and protein levels by RT-PCR and western blot assays, accordingly. Our results showed a significant expression of ALDH1L1 mRNA and protein expression in A549 cells carrying the Lv-ALDH1L1 vector. The lentivirus-induced ALDH1L1 elevation produced strong inhibitory effects on cellular proliferation and colony formation under nutrient-rich conditions. Of note, it also induced alterations of cell morphology. To explore the underlying mechanisms of these effects, apoptosis-related markers were examined. Elevated levels of the signal transduction pathways involved in initiating apoptosis; BID, BAX, and BAK in A549 cells transduced with Lv-ALDH1L1 were observed. Furthermore, using the wound healing assay, we showed that ALDH1L1 strongly suppresses the migratory ability of A549 cells. Interestingly, this effect was associated with a robust down-regulation of the actin depolymerizing factor cofilin and its phosphorylated form. Overall, this study demonstrated that lentiviral ALDH1L1 knock-in produces strong anti-tumorigenesis effects in A549 cells. This further suggests that ALDH1L1 should be investigated as a therapeutic drug target in cancer treatment. Citation Format: Amira Abdellatef, Sergey Krupenko. Lentivirus-induced ALDH1L1 knock-in inhibits proliferation and migration and activates apoptosis of human A549 lung adenocarcinoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B03.

Abstract LB_B09: Loss of folate enzyme ALDH1L1 promotes cancer cell proliferation and xenograft tumor growth

Abstract Background: ALDH1L1 (aldehyde dehydrogenase 1 family member L1, or cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme in folate metabolism, is abundantly expressed in several tissues. This enzyme converts 10-formyltetrahydrofolate to tetrahydrofolate (THF) and CO2 while reducing NADP+ to NADPH. ALDH1L1 is ubiquitously downregulated human cancers through DNA promoter methylation, while one cancer cell line, RT4 (derived from papillary bladder cancer), maintains high expression of ALDH1L1. Aims: We tested the hypothesis that loss of ALDH1L1 expression could provide a proliferative and survival advantage for cancer cells and tumor growth. Methods: The ALDH1L1 gene in RT4 cells was knocked down using the shRNA technique. Proliferation was evaluated using MTT assays and xCELLigence real-time cellular analysis (RTCA). Cell migration was assessed by wound healing assays. NADP+/NADPH levels and reduced folate pools were measured by ELISA and the ternary complex assays, respectively. RT4 tumors were generated in nude mice from RT4 lines by subcutaneous injections into both flanks of athymic nude mice. Tumor growth was monitored every week for 12-weeks, after which the tumors were harvested. ALDH1L1 levels in tumors were measured by Western Blot assays, while tumor proliferation was assessed by the Ki-67/H&E staining of the tumor tissue sections. Results: We found that doubling time did not differ between the wild-type (RT4) and shControl (shC) cell lines. Doubling time for the sh506 and sh572 clones was shorter than the controls by 68% and 79%, respectively. Proliferation and migration were significantly increased in sh506 and sh572 cells compared to the control lines. NADP+/NADPH levels were significantly increased for only the sh572 clone. Interestingly, tumor volume and weight were increased in both sh506 and sh572 derived tumors compared to shC tumors. The sh506 and sh572 tumors also showed a slightly increased Ki-67 proliferation index. The ALDH1L1 protein expression was completely lost in sh572 tumors but only partially decreased in sh506 tumors. Conclusions: Our study indicates that RT4 clones lacking ALDH1L1 have increased cell proliferation and tumor growth compared to wild-type RT4 cells, supporting the role of ALDH1L1 as a metabolic regulator of proliferation. Citation Format: Halle M Fogle, Mikyoung You, Jaspreet Sharma, Jonathan McCormac, Natalia I Krupenko, Sergey A Krupenko. Loss of folate enzyme ALDH1L1 promotes cancer cell proliferation and xenograft tumor growth [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B09.

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

Cerebral Folate Metabolism in Post-Mortem Alzheimer’s Disease Tissues: A Small Cohort Study

We investigated the cerebral folate system in post-mortem brains and matched cerebrospinal fluid (CSF) samples from subjects with definite Alzheimer's disease (AD) (n = 21) and neuropathologically normal brains (n = 21) using immunohistochemistry, Western blot and dot blot. In AD the CSF showed a significant decrease in 10-formyl tetrahydrofolate dehydrogenase (FDH), a critical folate binding protein and enzyme in the CSF, as well as in the main folate transporter, folate receptor alpha (FRα) and folate. In tissue, we found a switch in the pathway of folate supply to the cerebral cortex in AD compared to neurologically normal brains. FRα switched from entry through FDH-positive astrocytes in normal, to entry through glial fibrillary acidic protein (GFAP)-positive astrocytes in the AD cortex. Moreover, this switch correlated with an apparent change in metabolic direction to hypermethylation of neurons in AD. Our data suggest that the reduction in FDH in CSF prohibits FRα-folate entry via FDH-positive astrocytes and promotes entry through the GFAP pathway directly to neurons for hypermethylation. This data may explain some of the cognitive decline not attributable to the loss of neurons alone and presents a target for potential treatment.

Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

Abnormal downregulation of 10-formyltetrahydrofolate dehydrogenase promotes the progression of oral squamous cell carcinoma by activating PI3K/Akt/Rb pathway.

10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is a major folate enzyme, which is usually underexpressed in malignant tumors and competes with tumors for the same folate substrate. However, the specific role and mechanisms of ALDH1L1 in oral squamous cell carcinoma (OSCC) remainsobscure. The expression level of ALDH1L1 in paired OSCC tissues and adjacent noncancerous tissues were detected by quantitative realtime PCR, Western blot and immunohistochemistry. The relationship between ALDH1L1 expression and clinical characteristics was analyzed. Besides, CCK8, EdU staining, colony formation, wound healing, transwell invasion, apoptosis, cell cycle assays and nude mice tumor bearing experiments were employed to assess the role of ALDH1L1 in OSCC. To explore the underlying mechanisms of these effects, cell cycle-related markers were examined. In this study, we revealed that ALDH1L1 expression was significantly reduced in OSCC, and its downregulation was associated with the malignancy of the tumor and poor prognosis of patients. In vivo and in vitro experiments, downregulation of ALDH1L1 in OSCC significantly inhibited the occurrence of NADP+ -dependent catalytic reactions and facilitated tumor cell growth, migration, invasion, survival, cell cycle progression, and xenograft tumor growth. On the contrary, re-expression of ALDH1L1 plays a similar role to anti-folate therapy, promoting NADPH production and suppressing the progression of OSCC. Furthermore, ALDH1L1 overexpressing obviously inhibited the expression of PI3K, p-Akt, CDK2, CDK6, Cyclin D1, Cyclin D3, and Rb in OSCC cells, and promoted the expression of p27. LY294002 and 740 Y-P were used to confirm the inhibitory effects of ALDH1L1 on OSCC progression through PI3K/Akt/Rb pathway. Our findings highlight the clinical value of ALDH1L1 as a prognostic marker and the potential of a new target for anti-folate therapy.

Transcriptome analysis of hepatopancreas and gills of Palaemon gravieri under salinity stress

Salinity is an important factor in the aquatic environment, and its fluctuations always result in osmotic stress, which affects the survival, distribution, and physiological activities of crustaceans. Crustaceans counter them through osmoregulation, which consists of many mechanisms. Palaemon gravieri is an important economic species in Palaemonidae, widely distributed in the southern East China Sea and the China Yellow Sea, and has a good adaptability to salinity stress. Currently, there are only a few studies on the effects of salinity on P. graviera. Therefore, it is particularly important to study the molecular responses of P. gravieri to salinity fluctuations. In this study, P. gravieri was treated with salinities of 10, 25, and 40, and the hepatopancreas and gills of shrimp in the different salinity groups were sampled after 24 h. The samples were used for RNA extraction and transcriptome analysis. In total, 80,994 unigenes were obtained, of which 19,114 were annotated. The differences in gene expression between different tissues at the same salinity were more significant. Many metabolism-related genes were downregulated in the gills, such as beta-hexosaminidase subunit alpha (HEXA), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and Alcohol dehydrogenase class-3 (ADH5). Scanning transmission electron microscope analysis showed that the expression levels of some stress-(but not salinity stress) related genes changed after stress (mostly upregulated), suggesting the existence of secondary stress. Gene set enrichment analysis (GSEA) focused on the expression of transporters in osmoregulation, and the results showed that they mainly played a role in the gills, but ATP-binding cassette (ABC) transporters were more active in the hepatopancreas. This study showed that the response of P. gravieri to salinity change was different not only between the hepatopancreas and gills, but also between low salinity and higher salinity, and the ion transport-related genes were mainly expressed in the gills. Overall, these results improve our understanding of salt tolerance mechanism in P. gravieri.

Genetic variants in ALDH1L1 and GLDC influence the serine-to-glycine ratio in Hispanic children

Glycine is a proteogenic amino acid that is required for numerous metabolic pathways, including purine, creatine, heme, and glutathione biosynthesis. Glycine formation from serine, catalyzed by serine hydroxy methyltransferase, is the major source of this amino acid in humans. Our previous studies in a mouse model have shown a crucial role for the 10-formyltetrahydrofolate dehydrogenase enzyme in serine-to-glycine conversion. We sought to determine the genomic influence on the serine-glycine ratio in 803 Hispanic children from 319 families of the Viva La Familia cohort. We performed a genome-wide association analysis for plasma serine, glycine, and the serine-glycine ratio in Sequential Oligogenic Linkage Analysis Routines while accounting for relationships among family members. All 3 parameters were significantly heritable (h2=0.22-0.78; P<0.004). The strongest associations for the serine-glycine ratio were with single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase 1 family member L1 (ALDH1L1) and glycine decarboxylase (GLDC) and for glycine with GLDC (P<3.5×10-8; effect sizes, 0.03-0.07). No significant associations were found for serine. We also conducted a targeted genetic analysis with ALDH1L1 exonic SNPs and found significant associations between the serine-glycine ratio and rs2886059 (β = 0.68; SE, 0.25; P=0.006) and rs3796191 (β = 0.25; SE, 0.08; P=0.003) and between glycine and rs3796191 (β = -0.08; SE, 0.02; P=0.0004). These exonic SNPs were further associated with metabolic disease risk factors, mainly adiposity measures (P<0.006). Significant genetic and phenotypic correlations were found for glycine and the serine-glycine ratio with metabolic disease risk factors, including adiposity, insulin sensitivity, and inflammation-related phenotypes [estimate of genetic correlation = -0.37 to 0.35 (P<0.03); estimate of phenotypic correlation = -0.19 to 0.13 (P<0.006)]. The significant genetic correlations indicate shared genetic effects among glycine, the serine-glycine ratio, and adiposity and insulin sensitivity phenotypes. Our study suggests that ALDH1L1 and GLDC SNPs influence the serine-to-glycine ratio and metabolic disease risk.

Sex-Specific Metabolic Effects of Dietary Folate Withdrawal in Wild-Type and Aldh1l1 Knockout Mice.

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in the liver. It regulates the overall flux of folate-bound one-carbon groups by converting 10-formyltetrahydrofolate to tetrahydrofolate and CO2 in a NADP+-dependent reaction. Our previous study revealed that Aldh1l1 knockout (KO) mice have an altered liver metabotype with metabolic symptoms of folate deficiency when fed a standard chow diet containing 2 ppm folic acid. Here we performed untargeted metabolomic analysis of liver and plasma of KO and wild-type (WT) male and female mice fed for 16 weeks either standard or folate-deficient diet. OPLS-DA, a supervised multivariate technique that was applied to 6595 and 10,678 features for the liver and plasma datasets, respectively, indicated that genotype and diet, alone or in combination, gave distinct metabolic profiles in both types of biospecimens. A more detailed analysis of affected metabolic pathways based on most confidently identified metabolites in the liver and plasma (OL1 and OL2a ontology level) indicated that the dietary folate restriction itself does not fully recapitulate the metabolic effect of the KO. Of note, dietary folate withdrawal enhanced the metabolic perturbations linked to the ALDH1L1 loss only for a subset of metabolites. Importantly, both the ALDH1L1 loss and dietary folate deficiency produced sex-specific metabolic effects.

A Detoxification Enzyme for Apis mellifera Newly Characterized by Recombinant Expression: 10-Formyl Tetrahydrofolate Dehydrogenase.

Honeybees are important managed pollinators that perform important ecological and economic functions. In recent decades, the obligate ectoparasite Varroa destructor severely affected survival of honeybees as it either feeds on hemolymph and fat bodies or acts as a vector for viruses. A common treatment against the varroa mite is formic acid, which has been used for many years by beekeepers. This treatment is known to be effective, but the therapeutic index is very narrow. Many beekeepers report negative effects of formic acid on bees, which include damage to brood, worker bee mortality, and queen loss. Little is yet known about the molecular mechanisms of formic acid detoxification in honeybees. Our previous study shows the upregulation of predicted 10-formyl tetrahydrofolate dehydrogenase (10-FTHFDH) transcripts in honeybees exposed to formic acid. Here, the predicted honeybee-specific 10-FTHFDH is recombinantly expressed, and its hydrolase and dehydrogenase activities are investigated. As a result, the enzyme shows similar dehydrogenase activity in comparison to known 10-FTHFDHs. This study provides further knowledge to better understand the detoxification mechanisms of formic acid in Apis mellifera.

Abstract 2257: Effect ofALDH1L1folate enzyme on hepatocellular carcinoma inachemical carcinogenesismodel

Abstract Background: ALDH1L1 (aldehyde dehydrogenase 1 family member L1, or cytosolic 10-formyltetrahydrofolate dehydrogenase) is an abundant enzyme in folate metabolism representing about 1% of total soluble protein in hepatocytes. Expression of this protein is strongly and ubiquitously downregulated in human cancers through the promoter methylation. Several studies indicate that patients with hepatocellular carcinoma who had low expression of ALDH1L1 had poorer prognosis than those with high expression. Hypothesis: We propose that in the absence of ALDH1L1, liver tumors proliferate faster and grow to a larger size. Methods: The effect of the Aldh1l1 knockout (Aldh1l1−/−) on hepatocellular carcinoma was investigated using DEN (diethyl nitrosamine) model of chemical carcinogenesis. Fifteen-days-old male Aldh1l1−/- mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with DEN at the dose of 15 mg/kg body weight. Mice were sacrificed 10, 20, 28- and 36-weeks post-injection and livers were examined for for histopathology. Results: Significant reduction in total body weight was observed in the DEN-injected knockout compared to wild type animals at 28 and 36 weeks. Immunohistochemical analysis reveals that tumor size was significantly bigger in Aldh1l1−/− compared to Aldh1l1+/+ mice and also indicated fibrosis in livers of knockout vs wild-type mice. Vascular involvement, highlighted by reticulin staining, was present in both Aldh1l1−/− and Aldh1l1+/+ mice as early as 20 weeks after the injection. Tumor multiplicity did not differ significantly between the Aldh1l1−/− and Aldh1l1+/+ animals. Conclusion:. Aldh1l1 knockout promoted aggressiveness of liver tumors without affecting the tumor initiation. Acknowledgement: These studies were supported by the National Institutes of Health grants DK54388 and CA095030 (S.A.K.). Citation Format: Jaspreet Sharma, Natalia I. Krupenko, Peter Pediaditakis, Kristi Helke, Kyle C. Strickland, Sergey A. Krupenko. Effect ofALDH1L1folate enzyme on hepatocellular carcinoma inachemical carcinogenesismodel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2257.

Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis.

Simple SummaryCancers often loose the enzyme of folate metabolism ALDH1L1. We proposed that such loss is advantageous for the malignant tumor growth and tested this hypothesis in mice proficient or deficient (gene knockout) in ALDH1L1 expression. Liver cancer in both groups was induced by injection of chemical carcinogen diethylnitrosamine. While the number of tumors observed in ALDH1L1 proficient and deficient mice was similar, tumors grew faster and to a larger size in the knockout mice. We conclude that the ALDH1L1 loss promotes liver tumor growth without affecting tumor initiation or multiplicity. Accelerated growth of tumors lacking the enzyme was linked to several metabolic pathways, which are beneficial for rapid proliferation.Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1−/−) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 μg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1−/− and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1−/− compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1−/− and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1−/− metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

Metabolic Effects of ALDH1L1 Knockout in Diethylnitrosamine‐Induced Model of Liver Carcinogenesis

Background Global gene expression profiling identified ALDH1L1 (aldehyde dehydrogenase 1 family member L1, or cytosolic 10-formyltetrahydrofolate dehydrogenase) as one of the most under expressed genes in HCCs compared to normal liver (1). ALDH1L1 is an abundant enzyme in folate metabolism representing about 1% of total soluble protein in hepatocytes. Expression of this protein is strongly and ubiquitously downregulated in human cancers through the promoter methylation. Aim The goal of this study was to analyze changes in metabolic profiles of mouse livers depending on ALDH1L1 status in a diethyl nitrosamine (DEN)-induced model of liver carcinogenesis. Methods Fifteen-days-old male Aldh1l1-/-mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with DEN at the dose of 15 mg/kg body weight. Individual liver samples were collected after 10- and 20-weeks following treatment with DEN and subjected to metabolomics analysis in equivalent manner across multiple analytical platforms. Results Principal component analysis (PCA) showed that the differences in metabotypes between KO and WT mice were more prominent at 20 weeks then at 10 weeks. Several major cellular pathways contributed to the separation between Aldh1l1+/+and Aldh1l1-/- mice in their metabolic responses to DEN including pathways of folate, glycine, lipids and carbohydrates. Additionally, changes in redox-active compounds showed increased oxidative stress/decreased antioxidant capacity in KO livers. Conclusion Metabolic changes in KO livers support enhanced cell proliferation and reveal how the ALDH1L1 loss provides metabolic advantage for rapidly proliferating cells. References Tackels-Horne, D.; Goodman, M.D.; Williams, A.J.; Wilson, D.J.; Eskandari, T.; Vogt, L.M.; Boland, J.F.; Scherf, U.; Vockley, J.G. Identification of differentially expressed genes in hepatocellular carcinoma and metastatic liver tumors by oligonucleotide expression profiling. Cancer 2001, 92, 395-405.

Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naïve and stimulated cultures.

Astrocytes are responsible for a wide variety of essential functions throughout the central nervous system. The protein markers glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), glutamine synthetase (GS), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and the transcription factor SOX9 are routinely used to label astrocytes in primary rodent cultures. However, GLAST, GLT-1, GS, and SOX9 are also produced by microglia and oligodendrocytes and GFAP, GLAST, GLT-1, and GS production levels are affected by astrocyte phenotypic changes associated with reactive astrogliosis. No group has performed a comprehensive immunocytochemical evaluation to quantify the percentage of cells labeled by these markers in vitro, nor compared changes in staining between cortex- and spinal cord-derived cells in naïve and stimulated cultures. Here, we quantified the percentage of cells positively stained for these six markers in astrocyte, microglia, and oligodendrocyte cultures isolated from neonatal rat cortices and spinal cords. Additionally, we incubated the astrocytes with transforming growth factor (TGF)-β1 or TGF-β3 to determine if the labeling of these markers is altered by these stimuli. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Furthermore, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, and ALDH1L1 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST and GS in TGF-β1-treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models.

Comparative transcriptomics indicates endogenous differences in detoxification capacity after formic acid treatment between honey bees and varroa mites

Formic acid (FA) has been used for decades to control Varroa destructor, one of the most important parasites of the western honey bee, Apis mellifera. The rather unselective molecular mode of action of FA and its possible effects on honeybees have long been a concern of beekeepers, as it has undesirable side effects that affect the health of bee colonies. This study focuses on short-term transcriptomic changes as analysed by RNAseq in both larval and adult honey bees and in mites after FA treatment under applied conditions. Our study aims to identify those genes in honey bees and varroa mites differentially expressed upon a typical FA hive exposure scenario. Five detoxification-related genes were identified with significantly enhanced and one gene with significantly decreased expression under FA exposure. Regulated genes in our test setting included members of various cytochrome P450 subfamilies, a flavin-dependent monooxygenase and a cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH), known to be involved in formate metabolism in mammals. We were able to detect differences in the regulation of detoxification-associated genes between mites and honey bees as well as between the two different developmental stages of the honey bee. Additionally, we detected repressed regulation of Varroa genes involved in cellular respiration, suggesting mitochondrial dysfunction and supporting the current view on the mode of action of FA—inhibition of oxidative phosphorylation. This study shows distinct cellular effects induced by FA on the global transcriptome of both host and parasite in comparison. Our expression data might help to identify possible differences in the affected metabolic pathways and thus make a first contribution to elucidate the mode of detoxification of FA.

Proteomic changes by radio-mitigative thrombopoietin receptor agonist romiplostim in the blood of mice exposed to lethal total-body irradiation

Purpose The thrombopoietin receptor agonist romiplostim (RP) is a therapeutic agent for immune thrombocytopenia that can achieve complete survival in mice exposed to a lethal dose of ionizing radiation. The estimated mechanism of the radio-protective/mitigative effects of RP has been proposed; however, the detailed mechanism of action remains unclear. This study aimed to elucidate the mechanism of the radio-protective/mitigative effects of RP, the fluctuation of protein in the blood was analyzed by proteomics. Materials and Methods Eight-week-old female C57BL/6J mice were randomly divided into 5 groups; control at day 0, total-body irradiation (TBI) groups at day 10 and day 18, and TBI plus RP groups at day 10 and day18, consisting of 3 mice per group, and subjected to TBI with 7 Gy of 137Cs γ-rays at a dose rate of 0.74 Gy/min. RP was administered intraperitoneally to mice at a dose of 50 µg/kg once daily for 3 days starting 2 hours after TBI. On day 10 and day 18 after TBI, serum collected from each mouse was analyzed by liquid chromatography tandem mass spectrometry. Results Nine proteins were identified by proteomics methods from 269 analyzed proteins detected in mice exposed to a lethal dose of TBI: keratin, type II cytoskeletal 1 (KRT1), fructose-1, 6-bisphosphatase (FBP1), cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), peptidyl-prolyl cis-trans isomerase A (PPIA), glycine N-methyltransferase (GNMT), glutathione S-transferase Mu 1 (GSTM1), regucalcin (RGN), fructose-bisphosphate aldolase B (ALDOB) and betain–homocysteine S-methyltransferase 1 (BHMT). On the 10th day after TBI, KRT1 was significantly increased (p < 0.05) by 4.26-fold compared to the control group in the TBI group and significantly inhibited in the TBI plus RP group (p < 0.05). Similarly, the expression levels of other 8 proteins detected at 18th day after TBI were significantly increased by 4.29 to 27.44-fold in the TBI group, but significantly decreased in the TBI plus RP group compared to the TBI group, respectively. Conclusion Nine proteins were identified by proteomics methods from 269 analyzed proteins detected in mice exposed to a lethal dose of TBI. These proteins are also expected to be indicators of the damage induced by high-dose radiation.

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver

ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

Deleterious mutations in ALDH1L2 suggest a novel cause for neuro-ichthyotic syndrome

Neuro-ichthyotic syndromes are a group of rare genetic diseases mainly associated with perturbations in lipid metabolism, intracellular vesicle trafficking, or glycoprotein synthesis. Here, we report a patient with a neuro-ichthyotic syndrome associated with deleterious mutations in the ALDH1L2 (aldehyde dehydrogenase 1 family member L2) gene encoding for mitochondrial 10-formyltetrahydrofolate dehydrogenase. Using fibroblast culture established from the ALDH1L2-deficient patient, we demonstrated that the enzyme loss impaired mitochondrial function affecting both mitochondrial morphology and the pool of metabolites relevant to β-oxidation of fatty acids. Cells lacking the enzyme had distorted mitochondria, accumulated acylcarnitine derivatives and Krebs cycle intermediates, and had lower ATP and increased ADP/AMP indicative of a low energy index. Re-expression of functional ALDH1L2 enzyme in deficient cells restored the mitochondrial morphology and the metabolic profile of fibroblasts from healthy individuals. Our study underscores the role of ALDH1L2 in the maintenance of mitochondrial integrity and energy balance of the cell, and suggests the loss of the enzyme as the cause of neuro-cutaneous disease.

ALDH1L1 and ALDH1L2 Folate Regulatory Enzymes in Cancer.

Epidemiological studies implicate excess ethanol ingestion as a risk factor for several cancers and support the concept of a synergistic effect of chronic alcohol consumption and folate deficiency on carcinogenesis. Alcohol consumption affects folate-related genes and enzymes including two major folate-metabolizing enzymes, ALDH1L1 and ALDH1L2. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase) is a regulatory enzyme in folate metabolism that controls the overall flux of one-carbon groups in folate-dependent biosynthetic pathways. It is strongly and ubiquitously down-regulated in malignant tumors via promoter methylation, and recent studies underscored this enzyme as a candidate tumor suppressor and potential marker of aggressive cancers. A related enzyme, ALDH1L2, is the mitochondrial homolog of ALDH1L1 encoded by a separate gene. In contrast to its cytosolic counterpart, ALDH1L2 is expressed in malignant tumors and cancer cell lines and was implicated in metastasis regulation. This review discusses the link between folate and cancer, modifying effects of alcohol consumption on folate-associated carcinogenesis, and putative roles of ALDH1L1 and ALDH1L2 in this process.