The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA. We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project. To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells. To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors. To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair. Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1). In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. Disclosures Sridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.

Potential adverse health effects of dietary lipid oxidation products

Lipid oxidation products (LOPs) are widely present in many lipid- containing foods. They usually enter the gastrointestinal tract from dietary sources and/or are produced in vivo. Part of LOPs were absorbed into the blood and transported into tissues. A growing bulk of evidence suggests that LOPs, mainly reactive aldehydes and oxysterols, are potentially involved in the pathogenesis of many chronic diseases, such as atherosclerosis, Alzheimer’s disease, and inflammatory bowel disease. This review summarizes the current knowledge of the adverse effects, cytotoxicity, and the main mechanisms of LOPs involvement in humans and animals.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

Overview of the Neuroprotective Effects of the MAO-Inhibiting Antidepressant Phenelzine

Phenelzine (PLZ) is a monoamine oxidase (MAO)-inhibiting antidepressant with anxiolytic properties. This multifaceted drug has a number of pharmacological and neurochemical effects in addition to inhibition of MAO, and findings on these effects have contributed to a body of evidence indicating that PLZ also has neuroprotective/neurorescue properties. These attributes are reviewed in this paper and include catabolism to the active metabolite β-phenylethylidenehydrazine (PEH) and effects of PLZ and PEH on the GABA-glutamate balance in brain, sequestration of reactive aldehydes, and inhibition of primary amine oxidase. Also discussed are the encouraging findings of the effects of PLZ in animal models of stroke, spinal cord injury, traumatic brain injury, and multiple sclerosis, as well other actions such as reduction of nitrative stress, reduction of the effects of a toxin on dopaminergic neurons, potential anticonvulsant actions, and effects on brain-derived neurotrophic factor, neural cell adhesion molecules, an anti-apoptotic factor, and brain levels of ornithine and N-acetylamino acids.

Stratification Strategies for Patients to Optimize the Effectiveness of Scavenging Biogenic Aldehydes: Towards a Neuroprotective Approach for Parkinson’s Disease

Parkinson’s disease (PD) is a clinically heterogeneous disorder with a multi-factorial pathology. Various molecular mechanisms are involved in the pathogenesis of PD, converging to oxidative stress and proteinopathy. The accumulation of reactive aldehydes (i.e., the dopamine metabolite DOPAL, lipid-peroxidation products, and advanced glycation end-products) has been reported in PD patients’ brains. Aldehydes easily react with primary amines such as lysine residues, which are involved in several regulatory processes in cells. Therefore, aldehyde adducts lead to severe consequences, including neuronal proteostasis, mitochondrial dysfunction, and cell death. In this review, we analyzed the scavenging role of amines toward toxic aldehydes in the brain. Interestingly, small molecules like metformin, rasagiline, hydralazine are already clinically available and used in the therapy for PD and other diseases. Hence, we propose to reevaluate this class of drugs as a disease-modifiers for PD, and we suggest that improved analysis of their pharmacology and bioavailability in the brain, together with a more precise patients stratification, should be considered before planning future clinical trials.

Toxicity of reactive carbonyl compounds to plants

In plants, environmental stresses result in oxidative stress, lipid peroxidation and the generation of reactive carbonyl aldehydes. Reactive carbonyl aldehydes are downstream products of reactive oxygen species which can be described as critical cell-damaging agents in plants under various environmental stresses. In this paper toxicity of reactive carbonyl aldehydes and its generation under stress conditions are discussed. Moreover, involvement of reactive carbonyl aldehydes in stress- induced damage to plants is demonstrated. Toxic effect of reactive aldehydes such as acrolein, malondialdehyde and crotonaldehyde in plants under different stresses and their high electrophilicity is also discussed. Increases in malondialdehyde was demonstrated in UV-C stressed plants as the result of carbonyl modified proteins. A malondialdehyde is one of the widely shown aldehyde, which can be demonstrated as an indicator of reactive oxygen species. Malondialdehyde isomerized to 3-hydroxyacrolein whereas it can be described as a dialdehyde. The article considers detrimental actions of reactive carbonyl aldehydes and their chemical properties as well as detoxification of reactive carbonyl aldehydes by multiple enzymes such as aldehyde dehydrogenase, aldehyde reductase, aldo-keto reductase and 2-alkenal reductase.

Advanced Glycation End Products: Formation, Role in Diabetic Complications, and Potential in Clinical Applications

Hyperglycemic conditions and disruptions to glucose-regulating pathways lead to increased formation of highly reactive aldehydes, methylglyoxal and glyoxal, which react with certain arginine and lysine residues in proteins to form advanced glycation end products (AGEs). These AGEs damage the integrity of the retinal vasculature predominantly through two mechanisms: non-receptor-mediated damage, which pertains to the interaction with extracellular matrix and its functional properties, and receptor-mediated damage through AGE interactions with their receptors (RAGE) on pericytes and Muller cells. Damage occurring between AGE and RAGE potentially generates reactive oxygen species, inflammatory cytokines, and growth factors. Both mechanisms result in increased permeability of endothelial tight junctions, and this increased permeability can lead to leaking and eventually ischemia. Once this ischemia becomes significant, neovascularization can occur, the hallmark of proliferative diabetic retinopathy. Current pharmaceutical studies have shown the potential of AGE inhibitors, such as aminoguanidine, in decreasing AGE production, thus minimizing its effects in hyperglycemic conditions. Other pharmaceutical interventions, such as Tanshinone IIA, aim to protect cells from the impacts of AGEs. Future research will not only continue to understand the properties of AGEs and their effects on diabetes and diabetic complications like diabetic retinopathy but will also explore how they impact other diseases.

Refinement of the Peroxidase Peptide Reactivity Assay and Prediction Model for Assessing Skin Sensitization Potential

A peptide reactivity assay with an activation component was developed for use in screening chemicals for skin sensitization potential. A horseradish peroxidase-hydrogen peroxide (HRP/P) oxidation system was incorporated into the assay for characterizing reactivity of hapten and pre-/prohapten sensitizers. The assay, named the Peroxidase Peptide Reactivity Assay (PPRA) had a predictive accuracy of 83% (relative to the local lymph node assay) with the original protocol and prediction model. However, apparent false positives attributed to cysteine depletion at relatively high chemical concentrations and, for some chemicals expected to react with the −NH2 group of lysine, little to no depletion of the lysine peptide were observed. To improve the PPRA, cysteine peptide reactions with and without HRP/P were modified by increasing the number of test concentrations and refining their range. In addition, removal of DL-dithiothreitol from the reaction without HRP/P increased cysteine depletion and improved detection of reactive aldehydes and thiazolines without compromising the assay’s ability to detect prohaptens. Modification of the lysine reaction mixture by changing the buffer from 0.1 M ammonium acetate buffer (pH 10.2) to 0.1 M phosphate buffer (pH 7.4) and increasing the level of organic solvent from 1% to 25% resulted in increased lysine depletion for known lysine reactive chemicals. Refinement of the prediction model improved the sensitivity, specificity, and accuracy for hazard identification. These changes resulted in significant improvement of the PPRA making it is a reliable method for predicting the skin sensitization potential of all chemicals, including pre-/prohaptens and directly reactive haptens.

Second hits exacerbate alcohol-related organ damage: an update

Chronic and excessive alcohol abuse cause direct and indirect detrimental effects on a wide range of body organs and systems and accounts for ~4% of deaths worldwide. Many factors influence the harmful effects of alcohol. This concise review presents newer insights into the role of select second hits in influencing the progression of alcohol-induced organ damage by synergistically acting to generate a more dramatic downstream biological defect. This review specifically addresses on how a lifestyle factor of high fat intake exacerbates alcoholic liver injury and its progression. This review also provides the mechanistic insights into how increasing matrix stiffness during liver injury promotes alcohol-induced fibrogenesis. It also discusses how hepatotropic viral (HCV, HBV) infections as well as HIV (which is traditionally not known to be hepatotropic), are potentiated by alcohol exposure to promote hepatotoxicity and fibrosis progression. Finally, this review highlights the impact of reactive aldehydes generated during alcohol and cigarette smoke coexposure impair innate antimicrobial defense and increased susceptibility to infections. This review was inspired by the symposium held at the 17th Congress of the European Society for Biomedical research on Alcoholism in Lille, France entitled ‘Second hits in alcohol-related organ damage’.