Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production.

Pancreatic-islet inflammation contributes to the failure of ß cell insulin secretion during obesity and type 2 diabetes. However, little is known about the nature and function of resident immune cells in this context or in homeostasis. Here we show that interleukin (IL)-33 was produced by islet mesenchymal cells and enhanced by a diabetes milieu (glucose, IL-1ß, and palmitate). IL-33 promoted ß cell function through islet-resident group 2 innate lymphoid cells (ILC2s) that elicited retinoic acid (RA)-producing capacities in macrophages and dendritic cells via the secretion of IL-13 and colony-stimulating factor 2. In turn, local RA signaled to the ß cells to increase insulin secretion. This IL-33-ILC2 axis was activated after acute ß cell stress but was defective during chronic obesity. Accordingly, IL-33 injections rescued islet function in obese mice. Our findings provide evidence that an immunometabolic crosstalk between islet-derived IL-33, ILC2s, and myeloid cells fosters insulin secretion.

ALDH1+ tumor stem cells promote the progression of malignant fibrous tissue sarcoma by inhibiting SYNPO2 through hsa-mir-206.

This research aims to explore the mechanism by which microRNAs may regulate the biological behavior of tumor cells in ALDH1+ fibrosarcoma. We identified differentially expressed miRNAs in ALDH + NMFH-1 cells, screened genes related to sarcoma metastasis in the TCGA database, and finally obtained key genes regulated by miRNAs that are involved in metastasis. The function and mechanism of these key genes were then validated at the cellular level. Using the ULCAN database, a significant correlation was found between hsa-mir-206 and mortality in sarcoma patients. WGCNA analysis identified 352 genes related to tumor metastasis. Through Venn diagrams, we obtained 15 metastasis-related genes regulated by hsa-mir-206. Survival analysis showed that SYNPO2 expression is significantly correlated with survival rate and is significantly underexpressed in multiple tumors. SYNPO2 showed a negative correlation with macrophages and a positive correlation with CD8+ T cells. After inhibiting the expression of hsa-mir-206 with siRNA plasmids, the mRNA expression of SYNPO2 was significantly upregulated. The results of CCK8 assay, scratch assay, and transwell assay showed that the proliferation and migration ability of NFMH-1 cells were promoted after SYNPO2 was inhibited. ALDH1+ tumor stem cells promote the proliferation and invasion of malignant fibrous histiocytoma cells by inhibiting SYNPO2 through hsa-mir-206.

Acetylation of aldehyde dehydrogenase ALDH1L2 regulates cellular redox balance and the chemosensitivity of colorectal cancer to 5-fluorouracil.

Folate-mediated one-carbon metabolism (FOCM) is crucial in sustaining rapid proliferation and survival of cancer cells. The folate cycle depends on a series of key cellular enzymes, including aldehyde dehydrogenase 1 family member L2 (ALDH1L2) that is usually overexpressed in cancer cells, but the regulatory mechanism of ALDH1L2 remains undefined. In this study, we observed the significant overexpression of ALDH1L2 in colorectal cancer (CRC) tissues, which is associated with poor prognosis. Mechanistically, we identified that the acetylation of ALDH1L2 at the K70 site is an important regulatory mechanism inhibiting the enzymatic activity of ALDH1L2 and disturbing cellular redox balance. Moreover, we revealed that sirtuins 3 (SIRT3) directly binds and deacetylates ALDH1L2 to increase its activity. Interestingly, the chemotherapeutic agent 5-fluorouracil (5-Fu) inhibits the expression of SIRT3 and increases the acetylation levels of ALDH1L2 in colorectal cancer cells. 5-Fu-induced ALDH1L2 acetylation sufficiently inhibits its enzymatic activity and the production of NADPH and GSH, thereby leading to oxidative stress-induced apoptosis and suppressing tumor growth in mice. Furthermore, the K70Q mutant of ALDH1L2 sensitizes cancer cells to 5-Fu both in vitro and in vivo through perturbing cellular redox and serine metabolism. Our findings reveal an unknown 5-Fu-SIRT3-ALDH1L2 axis regulating redox homeostasis, and suggest that targeting ALDH1L2 is a promising therapeutic strategy to sensitize tumor cells to chemotherapeutic agents.

Further delineation of the phenotypic and metabolomic profile of ALDH1L2-related neurodevelopmental disorder.

ALDH1L2, a mitochondrial enzyme in folate metabolism, converts 10-formyl-THF (10-formyltetrahydrofolate) to THF (tetrahydrofolate) and CO2. At the cellular level, deficiency of this NADP+-dependent reaction results in marked reduction in NADPH/NADP+ ratio and reduced mitochondrial ATP. Thus far, a single patient with biallelic ALDH1L2 variants and the phenotype of a neurodevelopmental disorder has been reported. Here, we describe another patient with a neurodevelopmental disorder associated with a novel homozygous missense variant in ALDH1L2, Pro133His. The variant caused marked reduction in the ALDH1L2 enzyme activity in skin fibroblasts derived from the patient as probed by 10-FDDF, a stable synthetic analog of 10-formyl-THF. Additional associated abnormalities in these fibroblasts include reduced NADPH/NADP+ ratio and pool of mitochondrial ATP, upregulated autophagy and dramatically altered metabolomic profile. Overall, our study further supports a link between ALDH1L2 deficiency and abnormal neurodevelopment in humans.

Determination of pharmacological inhibition of ALDH2 by ethanol clearance in mice.

OBJECTIVES: Retinoic acid plays diverse physiological and pathophysiological roles in reproduction, immune function, energy metabolism and carcinogenesis. Because of the potential benefits of inhibiting retinoic acid biosynthesis in certain disease states, efforts are underway to develop inhibitors of retinoic acid biosynthesis via inhibition of the aldehyde dehydrogenase-1 A (ALDH1A) family of enzymes. However, many potential ALDH1A inhibitors also inhibit the related ALDH2 enzyme that plays a role in the metabolism of ethanol. Accurate in vitro assessment of ALDH2 inhibition is problematic, and to date, there are no published in vivo assays to determine inhibition of ALDH2 by candidate ALDH1A inhibitors. STUDY DESIGN: To address this, we developed a novel gas-chromatography-mass-spectrometry ethanol clearance assay in mice using orally administered ethanol and serial measurement of ethanol over time. We then used this assay to determine pharmacological inhibition of ALDH2 by candidate ALDH1A inhibitors. RESULTS: Ethanol clearance in untreated male mice occurs within sixty minutes. Male mice treated with WIN 18,446, a known ALDH1A inhibitor that also inhibits ALDH2, demonstrated significant inhibition of ethanol clearance compared to untreated controls. Novel pyrazole and piperazine ALDH1A inhibitors were then tested with the piperazine inhibitor demonstrating ALDH2 inhibition via impaired ethanol clearance while the pyrazole inhibitor did not interfere with ethanol metabolism, suggesting a lack of ALDH2 inhibition. CONCLUSIONS: Inhibition of ethanol clearance is a useful in vivo method of inferring pharmacologic inhibition of hepatic ALDH2. This assay may be useful in the development of novel ALDH1A specific inhibitors for a variety of therapeutic indications.

FOXO1 induced fatty acid oxidation in hepatic cells by targeting ALDH1L2.

BACKGROUND AND AIM: Lipid metabolism disorder is the primary feature of numerous refractory chronic diseases. Fatty acid oxidation, an essential aerobic biological process, is closely related to the progression of NAFLD. The forkhead transcription factor FOXO1 has been reported to play an important role in lipid metabolism. However, the molecular mechanism through which FOXO1 regulates fatty acid oxidation remains unclear. METHODS: Transcriptomic analysis was performed to examine the cellular expression profile to determine the functional role of FOXO1 in HepG2 cells with palmitic acid (PA)-induced lipid accumulation. FOXO1-binding motifs at the promoter region of aldehyde dehydrogenase 1 family member L2 (ALDH1L2) were predicted via bioinformatic analysis and confirmed via luciferase reporter assay. Overexpression of ALDH1L2 was induced to recover the impaired fatty acid oxidation in FOXO1-knockout cells. RESULTS: Knockout of FOXO1 aggravated lipid deposition in hepatic cells. Transcriptomic profiling revealed that knockout of FOXO1 increased the expression of genes associated with fatty acid synthesis but decreased the expression of carnitine palmitoyltransferase1a (CPT1α) and adipose triglyceride lipase (ATGL), which contribute to fatty acid oxidation. Mechanistically, FOXO1 was identified as a transcription factor of ALDH1L2. Knockout of FOXO1 significantly decreased the protein expression of ALDH1L2 and CPT1α in vitro and in vivo. Furthermore, overexpression of ALDH1L2 restored fatty acid oxidation in FOXO1-knockout cells. CONCLUSION: The findings of this study indicate that FOXO1 modulates fatty acid oxidation by targeting ALDH1L2.

A functional variant of ALDH1A2 is associated with hand osteoarthritis in the Chinese population.

Genome-wide association study identified common variants within the ALDH1A2 gene as the susceptible loci of hand osteoarthritis (HOA) in UK and Iceland populations. Located in chromosome 15, ALDH1A2 encodes aldehyde dehydrogenase family 1 member A2, which is an enzyme that catalyses the synthesis of retinoic acid from retinaldehyde. Our purposes were to replicate the association of functional variant in ALDH1A2 with the development of HOA in the Chinese population. Variant rs12915901 of ALDH1A2 was genotyped in 872 HOA patients and 1223 healthy controls. Subchondral bone samples were collected from 40 patients who had undergone a trapeziectomy, and the tissue expression of ALDH1A2 was analysed. The chi-square analysis was used to compare the frequency of genotype and risk allele between the HOA cases and controls. The Student t test was used to compare the mRNA expression of ALDH1A2 between patients with genotype AA/AG and those with genotype GG. The frequency of genotype AA was significantly higher in HOA patients than in the controls (7.6% vs. 5.1%, p = .01). The frequency of allele A was significantly higher in the patients than in the controls (28.9% vs. 24.6%, p = .005). The mRNA expression of ALDH1A2 was 1.31-folds higher in patients with genotype GG than in the patients with genotype AA/AG (0.000617 ± 0.000231 vs. 0.000471 ± 0.000198, p = .04). Variant rs12915901 of ALDH1A2 contributed to the susceptibility of HOA in the Chinese population. Allele A of rs12915901 can add to the risk of HOA possibly via down-regulation of ALDH1A2 expression.

High expression of PKCλ and ALDH1A3 indicates a poor prognosis, and PKCλ is required for the asymmetric cell division of ALDH1A3-positive cancer stem cells in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

ALDH1A1 and ALDH1A3 paralogues of aldehyde dehydrogenase 1 control myogenic differentiation of skeletal muscle satellite cells by retinoic acid-dependent and -independent mechanisms.

ALDH1A1 and ALDH1A3 paralogues of aldehyde dehydrogenase 1 (ALDH1) control myogenic differentiation of skeletal muscle satellite cells (SC) by formation of retinoic acid (RA) and subsequent cell cycle adjustments. The respective relevance of each paralogue for myogenic differentiation and the mechanistic interaction of each paralogue within RA-dependent and RA-independent pathways remain elusive.We analysed the impact of ALDH1A1 and ALDH1A3 activity on myogenesis of murine C2C12 myoblasts. Both paralogues are pivotal factors in myogenic differentiation, since CRISPR/Cas9-edited single paralogue knock-out impaired serum withdrawal-induced myogenic differentiation, while successive recombinant re-expression of ALDH1A1 or ALDH1A3, respectively, in the corresponding ALDH1 paralogue single knock-out cell lines, recovered the differentiation potential. Loss of differentiation in single knock-out cell lines was restored by treatment with RA-analogue TTNPB, while RA-receptor antagonization by AGN 193109 inhibited differentiation of wildtype cell lines, supporting the idea that RA-dependent pathway is pivotal for myogenic differentiation which is accomplished by both paralogues.However, overexpression of ALDH1-paralogues or disulfiram-mediated inhibition of ALDH1 enzymatic activity not only increased ALDH1A1 and ALDH1A3 protein levels but also induced subsequent differentiation of C2C12 myoblasts independently from serum withdrawal, indicating that ALDH1-dependent myogenic differentiation relies on different cellular conditions. Remarkably, ALDH1-paralogue knock-out impaired the autophagic flux, namely autophagosome cargo protein p62 formation and LC3B-I to LC3B-II conversion, demonstrating that ALDH1-paralogues interact with autophagy in myogenesis. Together, ALDH1 paralogues play a crucial role in myogenesis by orchestration of complex RA-dependent and RA-independent pathways.

Evidence for RA-dependent meiosis onset in a turtle embryo.

Meiotic entry is one of the earliest sex determination events of the germ cell in higher vertebrates. Although advances in meiosis onset have been achieved in mammals, birds and fish, how this process functions in reptiles is largely unknown. In this study, we present the molecular analysis of meiosis onset and the role of retinoic acid (RA) in this process in the red-eared slider turtle. Our results using Stra8 as a pre-meiosis indicator show that in the female embryonic gonad, meiosis commitment starts around stage 19. Additionally, signals of the meiosis marker Sycp3 could be detected at stage 19 and become highly expressed by stage 23. No expression of these genes was detected in male embryonic gonads, suggesting the entry into meiosis prophase I was restricted to female embryonic germ cells. Notably, RA activity in fetal gonads is likely to be elevated in females than that in males, as evidenced by the higher expression of RA synthase Aldh1a1 and lower expression of RA-degrading enzyme Cyp26a1 in female gonads prior to meiotic entry. In addition, exogenous RA treatment induced the expression of Stra8 and Sycp3 in both sexes, whether in vivo or in vitro. Together, these results indicate that high levels of RA in the embryonic female gonads can lead to the initiation of meiosis in the turtle.

Liver regeneration and ethanol detoxification: A new link in YAP regulation of ALDH1A1 during alcohol-related hepatocyte damage.

Yes-associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self-renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic-plus-single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl4 ). To understand the role of this transcriptional coactivator in alcohol-related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno-associated virus (AAV8)-mediated deletion utilizing Cre recombinase. Yap1 hepatocyte-specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b+ inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl4 -induced liver damage. Analysis of whole-genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1, a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl4 -induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol-associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.

ALDH1A2-related disorder: A new genetic syndrome due to alteration of the retinoic acid pathway.

Aldehyde Dehydrogenase 1, Family Member A2 (ALDH1A2) is essential for the synthesis of retinoic acid from vitamin A. Studies in model organisms demonstrate a critical role for ALDH1A2 in embryonic development, yet few pathogenic variants are linked to congenital anomalies in humans. We present three siblings with multiple congenital anomaly syndrome linked to biallelic sequence variants in ALDH1A2. The major congenital malformations affecting these children include tetralogy of Fallot, absent thymus, diaphragmatic eventration, and talipes equinovarus. Upper airway anomalies, hypocalcemia, and dysmorphic features are newly reported in this manuscript. In vitro functional validation of variants indicated that substitutions reduced the expression of the enzyme. Our clinical and functional data adds to a recent report of biallelic ALDH1A2 pathogenic variants in two families with a similar constellation of congenital malformations. These findings provide further evidence for an autosomal recessive ALDH1A2-deficient recognizable malformation syndrome involving the diaphragm, cardiac and musculoskeletal systems.

Altered ethanol metabolism and increased oxidative stress enhance alcohol-associated liver injury in farnesoid X receptor-deficient mice.

BACKGROUND & AIMS: Pharmacological activation of farnesoid X receptor (FXR) ameliorates liver injury, steatosis and inflammation in mouse models of alcoholic liver disease (ALD), but the underlying mechanisms of the protective effect of FXR against ALD remain unclear. METHODS: To investigate the role of FXR in ALD, we used the NIAAA model of chronic plus binge ethanol feeding in FXR-deficient knockout (FXR KO) mice. RESULTS: Ethanol-mediated liver injury and steatosis were increased in FXR KO mice, while both WT and FXR KO mice consumed the same amount of alcohol. Ethanol feeding induced liver inflammation and neutrophil infiltration that were further increased in FXR KO mice. In addition, collagen accumulation and expression of profibrotic genes were markedly elevated in the liver of alcohol-fed FXR KO compared to wild-type mice, suggesting that ethanol-induced liver fibrosis is enhanced in the absence of FXR. Surprisingly, FXR KO mice showed reduced blood alcohol levels post-binge, while CYP2E1 and ALDH1A1 were upregulated compared to WT mice, suggesting that alcohol metabolism is altered in FXR KO mice. Notably, exacerbated liver injury in FXR KO mice was associated with increased oxidative stress. ALDH1A1 activity was upregulated in FXR-deficient mouse primary hepatocytes, contributing to reactive oxygen species (ROS) generation, in vitro. Finally, using an ALDH1A1 inhibitor, we showed that ALDH1A1 activity is a key contributor to alcohol-induced ROS generation in FXR-deficient hepatocytes, in vitro. CONCLUSION: ALD pathogenesis in FXR KO mice correlates with altered ethanol metabolism and increased oxidative stress, providing new insights into the protective function of FXR in ALD.

Tumor heterogeneity from the viewpoint of pathologists.

Morphological and functional heterogeneity are found in tumors, with the latter reflecting the different levels of resistance against antitumor therapies. In a therapy-resistant subpopulation, the expression levels of differentiation markers decrease, and those of immature markers increase. In addition, this subpopulation expresses genes involved in drug metabolism, such as aldehyde dehydrogenase 1A1 (ALDH1A1). Because of their similarity to stem cells, cells in the latter therapy-resistant subpopulation are called cancer stem cells (CSCs). Like normal stem cells, CSCs were originally thought not to arise from non-CSCs, but this hierarchical model is too simple. It is now believed that CSCs are generated from non-CSCs. The plasticity of tumor phenotypes between CSCs and non-CSCs causes difficulty in completely curing tumors. In this review, focusing on ALDH1A1 as a marker for CSCs or immature tumor cells, the dynamics of ALDH1A1-expressing tumor cells and their regulatory mechanisms are described, and the plausible regulatory mechanisms of plasticity of ALDH1A1 expression phenotype are discussed. Genetic mutations are a significant factor for tumorigenesis, but non-mutational epigenetic reprogramming factors yielding tumor heterogeneity are also crucial in determining tumor characteristics. Factors influencing non-mutational epigenetic reprogramming in tumors are also discussed.

A Selective ALDH1A3 Inhibitor Impairs Mesothelioma 3-D Multicellular Spheroid Growth and Neutrophil Recruitment.

Aldehyde dehydrogenase 1A3 (ALDH1A3), one of the three members of the aldehyde dehydrogenase 1A subfamily, has been associated with increased progression and drug resistance in various types of solid tumours. Recently, it has been reported that high ALDH1A3 expression is prognostic of poor survival in patients with malignant pleural mesothelioma (MPM), an asbestos-associated chemoresistant cancer. We treated MPM cells, cultured as multicellular spheroids, with NR6, a potent and highly selective ALDH1A3 inhibitor. Here we report that NR6 treatment caused the accumulation of toxic aldehydes, induced DNA damage, CDKN2A expression and cell growth arrest. We observed that, in CDKN2A proficient cells, NR6 treatment induced IL6 expression, but abolished CXCL8 expression and IL-8 release, preventing both neutrophil recruitment and generation of neutrophil extracellular traps (NETs). Furthermore, we demonstrate that in response to ALDH1A3 inhibition, CDKN2A loss skewed cell fate from senescence to apoptosis. Dissecting the role of ALDH1A3 isoform in MPM cells and tumour microenvironment can open new fronts in the treatment of this cancer.

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.

The Molecular Context of Oxidant Stress Response in Cancer Establishes ALDH1A1 as a Critical Target: What This Means for Acute Myeloid Leukemia.

The protein family of aldehyde dehydrogenases (ALDH) encompasses nineteen members. The ALDH1 subfamily consists of enzymes with similar activity, having the capacity to neutralize lipid peroxidation products and to generate retinoic acid; however, only ALDH1A1 emerges as a significant risk factor in acute myeloid leukemia. Not only is the gene ALDH1A1 on average significantly overexpressed in the poor prognosis group at the RNA level, but its protein product, ALDH1A1 protects acute myeloid leukemia cells from lipid peroxidation byproducts. This capacity to protect cells can be ascribed to the stability of the enzyme under conditions of oxidant stress. The capacity to protect cells is evident both in vitro, as well as in mouse xenografts of those cells, shielding cells effectively from a number of potent antineoplastic agents. However, the role of ALDH1A1 in acute myeloid leukemia has been unclear in the past due to evidence that normal cells often have higher aldehyde dehydrogenase activity than leukemic cells. This being true, ALDH1A1 RNA expression is significantly associated with poor prognosis. It is hence imperative that ALDH1A1 is methodically targeted, particularly for the acute myeloid leukemia patients of the poor prognosis risk group that overexpress ALDH1A1 RNA.

Local and CNS-Wide Astrocyte Intracellular Calcium Signaling Attenuation In Vivo with CalExflox Mice.

Astrocytes exist throughout the CNS and affect neural circuits and behavior through intracellular Ca2+ signaling. Studying the function(s) of astrocyte Ca2+ signaling has proven difficult because of the paucity of tools to achieve selective attenuation. Based on recent studies, we generated and used male and female knock-in mice for Cre-dependent expression of mCherry-tagged hPMCA2w/b to attenuate astrocyte Ca2+ signaling in genetically defined cells in vivo (CalExflox mice for Calcium Extrusion). We characterized CalExflox mice following local AAV-Cre microinjections into the striatum and found reduced astrocyte Ca2+ signaling (∼90%) accompanied with repetitive self-grooming behavior. We also crossed CalExflox mice to astrocyte-specific Aldh1l1-Cre/ERT2 mice to achieve inducible global CNS-wide Ca2+ signaling attenuation. Within 6 d of induction in the bigenic mice, we observed significantly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality. Furthermore, with histologic, imaging, and transcriptomic analyses, we identified cellular and molecular alterations in the cerebellum following mCherry-tagged hPMCA2w/b expression. Our data show that expression of mCherry-tagged hPMCA2w/b with CalExflox mice throughout the CNS resulted in substantial attenuation of astrocyte Ca2+ signaling and significant behavioral alterations in adult mice. We interpreted these findings candidly in relation to the ability of CalEx to attenuate astrocyte Ca2+ signaling, with regards to additional mechanistic interpretations of the data, and their relation to past studies that reduced astrocyte Ca2+ signaling throughout the CNS. The data and resources provide complementary ways to interrogate the function(s) of astrocytes in multiple experimental scenarios.SIGNIFICANCE STATEMENT Astrocytes represent a significant fraction of all brain cells and tile the entire central nervous system. Unlike neurons, astrocytes lack propagated electrical signals. Instead, astrocytes are proposed to use diverse and dynamic intracellular Ca2+ signals to communicate with other cells. An open question concerns if and how astrocyte Ca2+ signaling regulates behavior in adult mice. We approached this problem by generating a new transgenic mouse line to achieve inducible astrocyte Ca2+ signaling attenuation in vivo We report our data with this mouse line and we interpret the findings candidly in relation to past studies and within the framework of different mechanistic interpretations.

Overexpression of N-acetylglucosaminyltransferase V promotes human parotid gland acinar cell immortalization via the epidermal receptor activation.

The purpose of this study is to maintain the proliferation capability of human parotid gland acinar cells (ACs) in vitro to extend passage number and to study the mechanism that regulates AC stemness. N-acetylglucosaminyltransferase V (GnT-V) is the Golgi enzyme, and it has been reported that the ß1,6GlcNAc-branched N-linked glycans are associated with various cell behaviors. Therefore, we modify the gene expression of ACs by transfection of the GnT-V-overexpression plasmid, and we found that upregulation of GnT-V extensively increased ACs proliferation and stemness properties in ACs/GnT-V compared to ACs transfected with Mock plasmid. More importantly, we observed that high levels of GnT-V positively correlated with ALDH1A3 expression via increasing phosphorylation of cell surface receptors and activating the downstream signaling transduction. Hence, the current study suggested that GnT-V is a significant factor for cell immortalization in the ACs model by activating the EGFR/ERK/ALDH1A3 signaling pathway.