The Immunometabolic Gene N-Acetylglucosamine Kinase Is Uniquely Involved in the Heritability of Multiple Sclerosis Severity.

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology.

Origin, evolution, and diversification of inositol 1,4,5-trisphosphate 3-kinases in plants and animals.

BACKGROUND: In Eukaryotes, inositol polyphosphates (InsPs) represent a large family of secondary messengers and play crucial roes in various cellular processes. InsPs are synthesized through a series of pohophorylation reactions catalyzed by various InsP kinases in a sequential manner. Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K), one member of InsP kinase, plays important regulation roles in InsPs metabolism by specifically phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4) in animal cells. IP3Ks were widespread in fungi, plants and animals. However, its evolutionary history and patterns have not been examined systematically. RESULTS: A total of 104 and 31 IP3K orthologues were identified across 57 plant genomes and 13 animal genomes, respectively. Phylogenetic analyses indicate that IP3K originated in the common ancestor before the divergence of fungi, plants and animals. In most plants and animals, IP3K maintained low-copy numbers suggesting functional conservation during plant and animal evolution. In Brassicaceae and vertebrate, IP3K underwent one and two duplication events, respectively, resulting in multiple gene copies. Whole-genome duplication (WGD) was the main mechanism for IP3K duplications, and the IP3K duplicates have experienced functional divergence. Finally, a hypothetical evolutionary model for the IP3K proteins is proposed based on phylogenetic theory. CONCLUSION: Our study reveals the evolutionary history of IP3K proteins and guides the future functions of animal, plant, and fungal IP3K proteins.

A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer.

OBJECTIVE: Availability of multimodal treatment strategies, including targeted therapies and immunotherapies, have improved the survival of non-small cell lung carcinoma (NSCLC). However, some patients still progress or respond poorly due to inherent resistance, acquired resistance, or lack of druggable driver mutations. Sphingosine-1-phosphate (S1P) and receptor tyrosine kinase-like orphan receptor (ROR1/2) signaling pathways are activated during lung carcinogenesis. METHODS: In this study, we have evaluated the crosstalk of S1P and ROR1/2 signaling pathways in lung cancer cells. RESULTS: S1P treatment of lung cancer cells decreases ROR1 and ROR2 transcript levels. While treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raises ROR1 and ROR2. Furthermore, simultaneous inhibition of SphK1 along with ROR1 reduced the migration of lung cancer cells. CONCLUSION: These findings demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e, in the absence of one pathway, another pathway may take charge of the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Ganciclovir and maribavir cross-resistance revisited: Relative drug susceptibilities of canonical cytomegalovirus mutants.

Therapeutic use of maribavir for human cytomegalovirus infection has renewed attention to the extent of cross-resistance with ganciclovir as the existing standard therapy. Each drug selects in vivo for a characteristic set of resistance mutations in the viral UL97 kinase gene. To improve the calibration of relative susceptibilities to each drug, genetic variants at relevant UL97 codons were extensively phenotyped using the same baseline viral clone, cell culture conditions and growth readout. Ganciclovir-selected mutations at codons 460, 520, 592, 594, 595 and 603 conferred 2.8-fold (C603Y) to 12-fold (M460I) increases in ganciclovir 50% inhibitory concentrations (EC50) over wild type baseline, while conferring maribavir EC50 fold changes ranging from 0.21-fold (M460I) to 1.9-fold (A594V). Maribavir-selected mutations at codons 409, 411 and 480 conferred maribavir EC50 fold changes ranging from 17 (H411Y) to 210 (C480F), while conferring ganciclovir EC50 fold changes ranging from 0.7 (H411Y) to 2.3 (C480F). The P-loop substitution F342Y, selected by either drug, is confirmed to confer 4.7-fold and 6-fold increases in maribavir and ganciclovir EC50s respectively, and suggests this part of the ATP-binding domain of UL97 to be involved in moderate resistance to both drugs. The maribavir hypersensitivity of M460I and M460V may be advantageous.

Novel PNKP mutations associated with reduced DNA single-strand break repair and severe microcephaly, seizures, and developmental delay.

BACKGROUND: Microcephaly with early-onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3'-phosphatase (PNKP). METHODS: We have used whole genome sequencing and Sanger sequencing to identify disease-causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP-ribose immunofluorescence. RESULTS: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single-strand break repair, confirming that the mutated PNKP alleles are dysfunctional. CONCLUSION: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1-induced single-strand breaks, and most likely causes MCSZ in this patient.

Metabolic Enzyme SLC27A5 Regulates PIP4K2A pre-mRNA Splicing as a Noncanonical Mechanism to Suppress Hepatocellular Carcinoma Metastasis.

Solute carrier family 27 member 5, a key enzyme in fatty acid transport and bile acid metabolism in the liver, is frequently expressed in low quantities in patients with hepatocellular carcinoma, resulting in poor prognosis. However, it is unclear whether SLC27A5 plays non-canonical functions and regulates HCC progression. Here, an unexpected non-canonical role of SLC27A5 is reported: regulating the alternative splicing of mRNA to inhibit the metastasis of HCC independently of its metabolic enzyme activity. Mechanistically, SLC27A5 interacts with IGF2BP3 to prevent its translocation into the nucleus, thereby inhibiting its binding to target mRNA and modulating PIP4K2A pre-mRNA splicing. Loss of SLC27A5 results in elevated levels of the PIP4K2A-S isoform, thus positively regulating phosphoinositide 3-kinase signaling via enhanced p85 stability in HCC. SLC27A5 restoration by AAV-Slc27a5 or IGF2BP3 RNA decoy oligonucleotides exerts an inhibitory effect on HCC metastasis with reduced expression of the PIP4K2A-S isoform. Therefore, PIP4K2A-S may be a novel target for treating HCC with SLC27A5 deficiency.

A novel homozygous variant in PMVK is associated with enhanced IL1ß secretion and a hyper-IgD syndrome-like phenotype.

The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate-limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper-IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS-like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1ß, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants.

Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Drug Resistance Assessed in a Phase 3 Clinical Trial of Maribavir Therapy for Refractory or Resistant Cytomegalovirus Infection in Transplant Recipients.

BACKGROUND: This drug resistance analysis of a randomized trial includes 234 patients receiving maribavir and 116 receiving investigator-assigned standard therapy (IAT), where 56% and 24%, respectively, cleared cytomegalovirus DNA at week 8 (treatment responders). METHODS: Baseline and posttreatment plasma samples were tested for mutations conferring drug resistance in viral genes UL97, UL54, and UL27. RESULTS: At baseline, genotypic testing revealed resistance to ganciclovir, foscarnet, or cidofovir in 56% of patients receiving maribavir and 68% receiving IAT, including 9 newly phenotyped mutations. Among them, 63% (maribavir) and 21% (IAT) were treatment responders. Detected baseline maribavir resistance mutations were UL27 L193F (n = 1) and UL97 F342Y (n = 3). Posttreatment, emergent maribavir resistance mutations were detected in 60 (26%) of those randomized to maribavir, including 49 (48%) of 103 nonresponders and 25 (86%) of the 29 nonresponders where viral DNA initially cleared then rebounded while on maribavir. The most common maribavir resistance mutations were UL97 T409M (n = 34), H411Y (n = 26), and C480F (n = 21), first detected 26 to 130 (median 56) days after starting maribavir. CONCLUSIONS: Baseline maribavir resistance was rare. Drug resistance to standard cytomegalovirus antivirals did not preclude treatment response to maribavir. Rebound in plasma cytomegalovirus DNA while on maribavir strongly suggests emerging drug resistance. CLINICAL TRIALS REGISTRATION: NCT02931539.

Aberrant accumulation of ceramides in mitochondria triggers cell death by inducing autophagy in Arabidopsis.

Sphingolipids are membrane lipids and play critical roles in signal transduction. Ceramides are central components of sphingolipid metabolism that are involved in cell death. However, the mechanism of ceramides regulating cell death in plants remains unclear. Here, we found that ceramides accumulated in mitochondria of accelerated cell death 5 mutant (acd5), and expression of mitochondrion-localized ceramide kinase (ACD5) suppressed mitochondrial ceramide accumulation and the acd5 cell death phenotype. Using immuno-electron microscopy, we observed hyperaccumulation of ceramides in acer acd5 double mutants, which are characterized by mutations in both ACER (alkaline ceramidase) and ACD5 genes. The results confirmed that plants with specific ceramide accumulation exhibited localization of ceramides to mitochondria, resulting in an increase in mitochondrial reactive oxygen species production. Interestingly, when compared with the wild type, autophagy-deficient mutants showed stronger resistance to ceramide-induced cell death. Lipid profiling analysis demonstrated that plants with ceramide accumulation exhibited a significant increase in phosphatidylethanolamine levels. Furthermore, exogenous ceramide treatment or endogenous ceramide accumulation induces autophagy. When exposed to exogenous ceramides, an increase in the level of the autophagy-specific ubiquitin-like protein, ATG8e, associated with mitochondria, where it directly bound to ceramides. Taken together, we propose that the accumulation of ceramides in mitochondria can induce cell death by regulating autophagy.

PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies.

Expression of protein arginine methyltransferase 5 (PRMT5) is highly positively correlated to DNA damage repair (DDR) and DNA replication pathway genes in many types of cancer cells, including ovarian and breast cancer. In the current study, we investigated whether pharmacologic inhibition of PRMT5 downregulates DDR/DNA replication pathway genes and sensitizes cancer cells to chemotherapy and PARP inhibition. Potent and selective PRMT5 inhibitors significantly downregulate expression of multiple DDR and DNA replication genes in cancer cells. Mechanistically, PRMT5 inhibition reduces the presence of PRMT5 and H4R3me2s on promoter regions of DDR genes such as BRCA1/2, RAD51, and ATM. PRMT5 inhibition also promotes global alternative splicing changes. Our data suggest that PRMT5 inhibition regulates expression of FANCA, PNKP, and ATM by promoting exon skipping and intron retention. Combining C220 or PRT543 with olaparib or chemotherapeutic agents such as cisplatin demonstrates a potent synergistic interaction in breast and ovarian cancer cells in vitro. Moreover, combination of PRT543 with olaparib effectively inhibits the growth of patient-derived breast and ovarian cancer xenografts. Furthermore, PRT543 treatment significantly inhibits growth of olaparib-resistant tumors in vivo. These studies reveal a novel mechanism of PRMT5 inhibition and suggest beneficial combinatorial effects with other therapies, particularly in patients with tumors that are resistant to therapies dependent on DNA damage as their mechanism of action. SIGNIFICANCE: Patients with advanced cancers frequently develop resistance to chemotherapy or PARP inhibitors mainly due to circumvention and/or restoration of the inactivated DDR pathway genes. We demonstrate that inhibition of PRMT5 significantly downregulates a broad range of the DDR and DNA replication pathway genes. PRMT5 inhibitors combined with chemotherapy or PARP inhibitors demonstrate synergistic suppression of cancer cell proliferation and growth in breast and ovarian tumor models, including PARP inhibitor-resistant tumors.

The first Vietnamese patient who presented late onset of pantothenate kinase-associated neurodegeneration diagnosed by whole exome sequencing: A case report.

RATIONALE: Pantothenate kinase-associated neurodegeneration (PKAN), also called Hallervorden-Spatz syndrome, is a rare autosomal recessive disease associated with brain iron accumulation and characterized by progressive dystonia, dementia, and dysarthria symptoms. PKAN, caused by a defective pantothenate kinase 2 (PANK2) gene, is the most common neurodegeneration with a brain iron accumulation (NBIA) group. The "eye of the tiger" sign in the magnetic resonance imaging demonstrated a bilateral hyperintense signal in the basal ganglia region on T2-weighted images, which is a characteristic feature of the diagnosis. PKAN is classified into 2 main types. The early-onset type (classic type) with rapid progression is characterized by symptoms of gait impairment and dystonia leading to loss of ambulation in early childhood. In the later-onset type (atypical type), slow progression usually takes place in the second decade of life with symptoms of neurodegeneration, dystonia, dysarthria, rigidity, choreoathetosis, and motor impairment. Until now, PKAN patients have only been reported in a few countries in Asia such as China, Korea, India, Iran, Taiwan, and Thailand. PATIENT CONCERNS: Here we report the first case of PKAN in Vietnam. The patient had a late onset but the disease progresses rapidly with symptoms of dyskinesia, dysphagia, and difficulty speaking. DIAGNOSES: Pantothenate kinase-associated neurodegeneration. INTERVENTIONS: Whole exome sequencing was performed to identify heterozygous mutations in the PANK2 gene (NM_153638.4) (c.856C>T, p.Arg286Cys and c.1351C>T, p.Arg451Ter) that has been confirmed as the cause of the disease. OUTCOMES: In this study, the first Vietnamese patient with late-onset PKAN was diagnosed by the whole exome sequencing method. LESSONS: The patient's case marks an important milestone for the first case in Vietnam. The results of the study will provide a scientific basis for clinicians in the diagnosis and genetic counseling of patients.

Inositol polyphosphate multikinase modulates free fatty acids-induced insulin resistance in primary mouse hepatocytes.

Insulin resistance is a critical mediator of the development of nonalcoholic fatty liver disease (NAFLD). An excess influx of fatty acids to the liver is thought to be a pathogenic cause of insulin resistance and the development of NAFLD. Although elevated levels of free fatty acids (FFA) in plasma contribute to inducing insulin resistance and NAFLD, the molecular mechanism is not completely understood. This study aimed to determine whether inositol polyphosphate multikinase (IPMK), a regulator of insulin signaling, plays any role in FFA-induced insulin resistance in primary hepatocytes. Here, we show that excess FFA decreased IPMK expression, and blockade of IPMK decrease attenuated the FFA-induced suppression of protein kinase B (Akt) phosphorylation in primary mouse hepatocytes (PMH). Moreover, overexpression of IPMK prevented the FFA-induced suppression of Akt phosphorylation by insulin, while knockout of IPMK exacerbated insulin resistance in PMH. In addition, treatment with MG132, a proteasomal inhibitor, inhibits FFA-induced decrease in IPMK expression and Akt phosphorylation in PMH. Furthermore, treatment with the antioxidant N-acetyl cysteine (NAC) significantly attenuated the FFA-induced reduction of IPMK and restored FFA-induced insulin resistance in PMH. In conclusion, our findings suggest that excess FFA reduces IPMK expression and contributes to the FFA-induced decrease in Akt phosphorylation in PMH, leading to insulin resistance. Our study highlights IPMK as a potential therapeutic target for preventing insulin resistance and NAFLD.

Commentary on: The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells.

1,4,5-triphosphate 3-kinase A (ITPKA) was first described and characterized by Irvine et al. in 1986 and cloned by Takazawa et al. in 1990. It is one of the components of the Ca2+ and calmodulin signaling pathway and a substrate for cAMP-dependent kinase (PKA) and protein kinase C (PKC), and is mainly involved in the regulation of intracellular inositol polyphosphate signaling molecules. Through a series of studies, Sabine's team has found that ITPKA expression was up-regulated in a variety of cancer cells, and silencing ITPKA inhibited while overexpressing ITPKA promoted cancer cell migration in vitro and metastasis in vivo. The latest research from Sabine's team has demonstrated that in H1299 lung cancer cells, the mechanism by which ITPKA promoted migration and invasion was predominantly depending on the ability of binding to F-actin, which will induce cancer cells to form a tight flexible actin networks. Small molecule compounds targeting the IP3 kinase activity of ITPKA protein may only inhibit the migration and invasion of cancer cells caused by the enhanced ITPKA kinase activity under ATP stimulation, but not the cytoskeletal remodeling caused by the binding of ITPKA protein to F-actin and the driven migration and invasion of cancer cells. Therefore, targeted therapeutic strategy focusing on blocking the binding of ITPKA to F-actin is indispensable when designing the inhibitors targeting ITPKA protein.

Exacerbated response to oxidative stress in the Retinitis Pigmentosa CerklKD/KO mouse model triggers retinal degeneration pathways upon acute light stress.

The retina is particularly vulnerable to genetic and environmental alterations that generate oxidative stress and cause cellular damage in photoreceptors and other retinal neurons, eventually leading to cell death. CERKL (CERamide Kinase-Like) mutations cause Retinitis Pigmentosa and Cone-Rod Dystrophy in humans, two disorders characterized by photoreceptor degeneration and progressive vision loss. CERKL is a resilience gene against oxidative stress, and its overexpression protects cells from oxidative stress-induced apoptosis. Besides, CERKL contributes to stress granule-formation and regulates mitochondrial dynamics in the retina. Using the CerklKD/KO albino mouse model, which recapitulates the human disease, we aimed to study the impact of Cerkl knockdown on stress response and activation of photoreceptor death mechanisms upon light/oxidative stress. After acute light injury, we assessed immediate or late retinal stress response, by combining both omic and non-omic approaches. Our results show that Cerkl knockdown increases ROS levels and causes a basal exacerbated stress state in the retina, through alterations in glutathione metabolism and stress granule production, overall compromising an adequate response to additional oxidative damage. As a consequence, several cell death mechanisms are triggered in CerklKD/KO retinas after acute light stress. Our studies indicate that Cerkl gene is a pivotal player in regulating light-challenged retinal homeostasis and shed light on how mutations in CERKL lead to blindness by dysregulation of the basal oxidative stress response in the retina.

ATF3 regulates SPHK1 in cardiomyocyte injury via endoplasmic reticulum stress.

AIM: Endoplasmic reticulum (ER) stress is common in different human pathologies, including cardiac diseases. Sphingosine kinase-1 (SPHK1) represents an important player in cardiac growth and function. Nevertheless, its function in cardiomyocyte ER stress remains vague. This study sought to evaluate the mechanism through which SPHK1 might influence ER stress during myocardial infarction (MI). METHODS: MI-related GEO data sets were queried to screen differentially expressed genes. Murine HL-1 cells exposed to oxygen-glucose deprivation (OGD) and mice with MI were induced, followed by gene expression manipulation using short hairpin RNAs and overexpression vectors. The activating transcription factor 3 (ATF3) and SPHK1 expression was examined in cells and tissues. Cell counting kit-8, TUNEL, DHE, HE, and Masson's staining were conducted in vitro and in vivo. The inflammatory factor concentrations in mouse serum were measured using ELISA. Finally, the transcriptional regulation of SPHK1 by ATF3 was validated. RESULTS: ATF3 and SPHK1 were upregulated in vivo and in vitro. ATF3 downregulation reduced the SPHK1 transcription. ATF3 and SPHK1 downregulation increased the viability of OGD-treated HL-1 cells and decreased apoptosis, oxidative stress, and ER stress. ATF3 and SPHK1 downregulation narrowed the infarction area and attenuated myocardial fibrosis in mice, along with reduced inflammation in the serum and ER stress in the myocardium. In contrast, SPHK1 reduced the protective effect of ATF3 downregulation in vitro and in vivo. CONCLUSIONS: ATF3 downregulation reduced SPHK1 expression to attenuate cardiomyocyte injury in MI.

Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.