A novel inducible animal model for studying chronic plasmalogen deficiency associated with Alzheimer's disease.

Plasmalogens are vinyl-ether glycerophospholipids critical for the structure and function of neuronal membranes. Deficient plasmalogen levels are associated with neurodegenerative diseases, particularly Alzheimer's disease (AD), which has led to the hypothesis that plasmalogen deficiency might drive disease onset and progression. However, the lack of a suitable animal model with late-onset plasmalogen deficiency has prevented testing of this hypothesis. The goal of this project was therefore to develop and characterize a mouse model capable of undergoing a plasmalogen deficiency only in adulthood, mirroring the chronic decline thought to occur in AD. We report here the creation of a novel animal model containing a tamoxifen-inducible knockout of the Gnpat gene encoding the first step in the plasmalogen biosynthetic pathway. Tamoxifen treatment in adult animals resulted in a significant reduction of plasmalogens in both the circulation and tissues as early as four weeks. By four months, changes in behavior and nerve function were observed, with strong correlations between residual brain plasmalogen levels, hyperactivity, and latency. The model will be useful for further elucidating the role of plasmalogens in AD and evaluating plasmalogen therapies.

CSE triggers ferroptosis via SIRT4-mediated GNPAT deacetylation in the pathogenesis of COPD.

BACKGROUND: It is now understood that ferroptosis plays a significant role in the progression of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke extract (CSE). However, the mechanisms underlying this relationship remain largely unclear. METHODS: In this study, we established a COPD mouse model through exposure to cigarette smoke particulates, followed by H&E staining, analysis of bronchoalveolar lavage fluid, and immunohistochemistry assay. A549 cells were exposed to increasing concentrations of CSE, with the addition of the ferroptosis activator erastin or the inhibitor Fer-1. Cell viability, LDH (lactate dehydrogenase) release, inflammatory cytokines, total ROS (reactive oxygen species), and lipid ROS were measured using the corresponding assay kits. The acetylation level of GNPAT was determined through immunoprecipitation. We assessed the expression levels of molecules involved in plasmalogen biosynthesis (FAR1, AGPS, and GNPAT), GPX4, and SIRT4 using quantitative real-time PCR, western blot analysis, and immunofluorescence staining. RESULTS: CSE-induced lung tissue damage was initially observed, accompanied by oxidative stress, ferroptosis, and increased plasmalogen biosynthesis molecules (FAR1, AGPS, and GNPAT). CSE also induced ferroptosis in A549 cells, resulting in reduced cell viability, GSH, and GPX4 levels, along with increased LDH, ROS, MDA (malondialdehyde) levels, oxidized lipids, and elevated FAR1, AGPS, and GNPAT expression. Knockdown of GNPAT mitigated CSE-induced ferroptosis. Furthermore, we found that CSE regulated the acetylation and protein levels of GNPAT by modulating SIRT4 expression. Importantly, the overexpression of GNPAT countered the inhibitory effects of SIRT4 on ferroptosis. CONCLUSIONS: Our study revealed GNPAT could be deacetylated by SIRT4, providing novel insights into the mechanisms underlying the relationship between CSE-induced ferroptosis and COPD.

Neonatal rhizomelic chondrodysplasia punctata type 2 caused by a novel homozygous variant in the GNPAT gene.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare disorder (~1 in 100,000 live births) of faulty plasmalogen biosynthesis and defective peroxisomal metabolism. RCDP type 2 is specifically caused by glyceronephosphate O-acyltransferase (GNPAT) gene mutations and is inherited as an autosomal recessive trait. The disorder is characterized by skeletal abnormalities, distinctive facial features, intellectual disability, and respiratory distress. The case report describes a newborn baby with a dysmorphic facial appearance and skeletal abnormalities who was admitted to neonatal intensive care with respiratory distress. His parents were first cousins. The whole exome sequencing for this patient identified an interesting homozygous variant in the GNPAT gene [GNPAT (NM_014236.4):c.1602+1G>A (p.?), Chr1 (GRCh37):g.231408138G>A]. This case report aims to highlight the patient's clinical presentation with the variant and the whole exome sequencing, indicating the identification of a novel mutation in the GNPAT gene causing RCDP type 2.

Time-Dependent Analysis of Plasmalogens in the Hippocampus of an Alzheimer's Disease Mouse Model: A Role of Ethanolamine Plasmalogen.

Plasmalogens are alkenyl-acyl glycerophospholipids and decreased in post-mortem Alzheimer's disease (AD) brains. The aim of this study is to investigate the time-dependent changes of plasmalogens in the hippocampus of an AD model mouse (J20). Plasmalogen levels at 3, 6, 9, 12 and 15 months were analyzed by liquid-chromatography-targeted-multiplexed-selected-reaction-monitoring-tandem-mass-spectrometry (LC-SRM/MS). Reactive oxygen species (ROS) levels were evaluated using dichlorofluorescein diacetate (DCF-DA). Plasmalogen synthesizing enzyme glycerone-phosphate O-acyltransferase (GNPAT) and late endosome marker Rab7 levels were quantified by Western blotting. GNPAT localization, changes of neuronal and glial cell numbers were evaluated by immunostaining. Compared to wild-type mice (WT), total plasmalogen-ethanolamine, but not plasmalogen-choline levels, were increased at 9 months and subsequently decreased at 15 months in J20 mice. A principal component analysis of plasmalogen-ethanolamine species could separate WT and J20 mice both at 9 and 15 months. Both GNPAT and Rab7 protein were increased in J20 mice at 9 months, whereas GNPAT was decreased at 15 months. ROS levels were increased in J20 mice except for 9 months. Our results suggest that increased plasmalogen-ethanolamine could counteract ROS levels and contribute to the phagocytosis process in J20 mice at 9 months. Such results might indicate a transient protective response of plasmalogen-ethanolamine in AD conditions.

Genetic epidemiology approach to estimating birth incidence and current disease prevalence for rhizomelic chondrodysplasia punctata.

BACKGROUND: Rhizomelic chondrodysplasia punctata (RCDP) is an inherited ultra-rare disease which results in severely impaired physical and mental development. Mutations in one of five genes involved in plasmalogen biosynthesis have been reported to drive disease pathology. Estimates of disease incidence have been extremely challenging due to the rarity of the disorder, preventing an understanding of the unmet medical need. To address this, we have prepared a disease incidence and prevalence model based on genetic epidemiology approaches to estimate the total number of RCDP patients affected, and their demographic characteristics. RESULTS: Extraction of allelic frequencies for known and predicted pathogenic variants in PEX7, GNPAT, AGPS, FAR1, PEX5 (limited to the PTS2 domain encoding region) genes, from large-scale human genetic diversity datasets (TopMed and gnomAD) revealed the mutational landscape contributing to the RCDP patient population in the US and Europe. We computed genetic prevalence to derive birth incidence for RCDP and modeled the impact to life expectancy to obtain high confidence estimates of disease prevalence. Our population genetics-based model indicates PEX7 variants are expected to contribute to the majority of RCDP cases in both the US and Europe; closely aligning with clinical reports. Furthermore, this model provides estimates for RCDP subtypes due to mutations in other genes, including exceedingly rare subtypes. CONCLUSION: In total, the estimated number of RCDP patients in the US and the five largest European countries (UK, Germany, France, Italy and Spain) is between 516 and 847 patients, all under the age of 35 years old. This model provides a quantitative framework for better understanding the unmet medical need in RCDP, to help guide disease awareness and diagnosis efforts for this specific patient group.

A new GNPAT variant of foetal rhizomelic chondrodysplasia punctata.

BACKGROUND: Rhizomelic chondrodysplasia punctata (RCDP) is a clinical entity resulting from defects of peroxisomal metabolism whose clinical phenotype is characterized by rhizomelia, calcified foci in periarticular cartilage, coronal lesions of vertebral bodies, cataracts and severe cognitive delay. Usually, survival does not exceed the first decade of life. Transmission is autosomal recessive and is related to mutations in the PEX7, GNPAT or AGPS. METHODS: A detailed description of the prenatal ultrasound signs of RCDP found in two successive pregnancies in a consanguineous couple is reported. Molecular genetic investigations included the study of the coding regions and the exon-intron junctions of the GNPAT (high-throughput amplification and sequencing performed with Roche NimbleGen SeqCap Target kit on Illumina platform); the confirmation test was carried out by amplification and Sanger sequencing with automatic capillary sequencer. RESULTS: In addition to the typical prenatal ultrasound signs described in the literature in association with RCDP, the presence of prefrontal oedema, never previously described, has been detected in both pregnancies. Moreover, genetic investigations have found a new splicing variant c.924+1G>A of the homozygous GNPAT. CONCLUSION: The role of mutation in the GNPAT suggests a likely association with the clinical phenotype.

Dysregulated hepcidin response to dietary iron in male mice with reduced Gnpat expression.

Exome sequencing has identified the glyceronephosphate O-acyltransferase (GNPAT) gene as a genetic modifier of iron overload in hereditary hemochromatosis (HH). Subjects with HFE (Homeostatic Iron Regulator) p.C282Y mutations and the GNPAT p.D519G variant had more iron loading compared with subjects without the GNPAT variant. In response to an oral iron challenge, women with GNPAT polymorphisms loaded more iron as compared with women without polymorphisms, reinforcing a role for GNPAT in iron homeostasis. The aim of the present study was to develop and characterize an animal model of disease to further our understanding of genetic modifiers, and in particular the role of GNPAT in iron homeostasis. We generated an Hfe/Gnpat mouse model reminiscent of the patients previously studied and studied these mice for up to 26 weeks. We also examined the effect of dietary iron loading on mice with reduced Gnpat expression. Gnpat heterozygosity in Hfe knockout mice does not play a role in systemic iron homeostasis; Gnpat+/- mice fed a high-iron diet, however, had lower hepatic hepcidin (HAMP) mRNA expression, whereas they have significantly higher serum iron levels and transferrin saturation compared with wildtype (WT) littermates on a similar diet. These results reinforce an independent role of GNPAT in systemic iron homeostasis, reproducing in an animal model, the observations in women with GNPAT polymorphisms subjected to an iron tolerance test.

Increased frequency of GNPAT p.D519G in compound HFE p.C282Y/p.H63D heterozygotes with elevated serum ferritin levels.

Glyceronephosphate O-acyltransferase (GNPAT) p.D519G (rs11558492) was identified as a genetic modifier correlated with more severe iron overload in hemochromatosis through whole-exome sequencing of HFE p.C282Y homozygotes with extreme iron phenotypes. We studied the prevalence of p.D519G in HFE p.C282Y/p.H63D compound heterozygotes, a genotype associated with iron overload in some patients. Cases were Australian participants with elevated serum ferritin (SF) levels ≥300µg/L (males) and ≥200µg/L (females); subjects whose SF levels were below these cut-offs were designated as controls. Samples were genotyped for GNPAT p.D519G. We compared the allele frequency of the present subjects, with/without elevated SF, to p.D519G frequency in public datasets. GNPAT p.D519G was more prevalent in our cohort of p.C282Y/p.H63D compound heterozygotes with elevated SF (37%) than European public datasets: 1000G 21%, gnomAD 20% and ESP 21%. We conclude that GNPAT p.D519G is associated with elevated SF in Australian HFE p.C282Y/p.H63D compound heterozygotes.

Gnpat does not play an essential role in systemic iron homeostasis in murine model.

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat-/- ), Gnpat/Hfe double-knockout (Gnpat-/- Hfe-/- or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpatfl/fl ;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat+/+ ) and Gnpat-/- mice, between Hfe-/- and DKO mice, or between Gnpatfl/fl and Gnpatfl/fl ;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat-/- and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe-/- mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe-/- mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe-/- mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.

1q42.12q42.2 Deletion in a Child with Midline Defects and Hypoplasia of the Corpus Callosum.

Chromosome 1q42.12q42.2 deletions are documented as "disease causing" and show overlapping phenotypes depending on the genes involved in the deletion. In this report, we detected a 5.8-Mb deletion encompassing the chromosome 1q42.12q42.2 region in a 4-year-old boy with hypoplastic corpus callosum, epilepsy, developmental delay, microcephaly, cataract, cleft palate, and skeletal changes. The deletion was de novo. Genotype-phenotype correlations suggest that the major features of 1q42.12q42.2 microdeletion were attributed to the genes with a high probability of loss-of-function intolerance score in this deletion, namely LBR, ENAH, ACBD3, LIN9, ITPKB, CDC42BPA, ARF1, TAF5L, GALNT2, SPRTN, and EGLN1 along with GNPAT.

Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation.

Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer's disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto the Gnpat promoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation of Gnpat in the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism of Gnpat reduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases.SIGNIFICANCE STATEMENT Ether-type glycerophospholipids, plasmalogens (Pls), are reduced in the brain of Alzheimer disease (AD) patients. We found that inflammatory stimuli reduced Pls contents by downregulation of the Pls-synthesizing enzyme glycerone phosphate O-acyltransferase (Gnpat) through NF-κB-mediated recruitment of c-Myc onto the Gnpat promoter in both murine and human cell lines. Murine brains after systemic lipopolysaccharide, chronic stress, and aging, as well as triple-transgenic AD mice and postmortem human AD brain tissues all showed increased c-Myc and reduced Gnpat expression. Interestingly, knockdown of Gnpat itself activated NF-κB in glial cell lines and microglia in mouse cortex. Our findings provide a new insight into the mechanism of neuroinflammation and may help to develop a novel therapeutic approach for neurodegenerative diseases such as AD.

The role of genetic factors in patients with hepatocellular carcinoma and iron overload - a prospective series of 234 patients.

BACKGROUND & AIMS: Iron overload (IO) in HFE-related hereditary haemochromatosis is associated with increased risk of liver cancer. This study aimed to investigate the role of other genes involved in hereditary IO among patients with hepatocellular carcinoma (HCC). METHODS: Patients with HCC diagnosed in our institution were included in this prospective study. Those with ferritin levels ≥300 µg/L (males) or ≥200 µg/L (females) and/or transferrin saturation ≥50% (males) or ≥45% (females) had liver iron concentration (LIC) evaluated by MRI. HFE C282Y and H63D mutations were screened. Genetic analyses of genes involved in hereditary IO (HFE, HJV/HFE2, HAMP, TFR2, SLC40A1, GNPAT) were performed in patients with increased LIC. RESULTS: A total of 234 patients were included; 215 (92%) had common acquired risk factors of HCC (mainly alcoholism or chronic viral hepatitis). 119 patients had abnormal iron parameters. Twelve (5.1%) were C282Y homozygotes, three were compound C282Y/H63D heterozygotes. LIC was measured by MRI in 100 patients. Thirteen patients with a LIC>70 µmol/g were enrolled in further genetic analyses: two unrelated patients bore the HAMP:c.-153C>T mutation at the heterozygous state, which is associated with increased risk of IO and severe haemochromatosis. Specific haplotypes of SLC40A1 were also studied. CONCLUSIONS: Additional genetic risk factors of IO were found in 18 patients (7.7%) among a large series of 234 HCC patients. Screening for IO and the associated at-risk genotypes in patients who have developed HCC, is useful for both determining etiologic diagnosis and enabling family screening and possibly primary prevention in relatives.

Altered phospholipid molecular species and glycolipid composition in brain, liver and fibroblasts of Zellweger syndrome.

We studied the altered molecular species of lipids in brain and liver tissues, and fibroblasts from patients with Zellweger syndrome (ZS). ZS cerebellum samples contained a higher amount of sphingomyelin with shorter chain fatty acids compared to that in normal controls. The amount of phosphatidylethanolamine (PE) was less than half of that in controls, with the absence of the PE-type of plasmalogen. Gangliosides were accumulated in the brains and fibroblasts of ZS patients. To investigate whether or not impaired beta-oxidation of very long chain fatty acids and/or plasmalogen synthesis affects glycolipids metabolism, RNAi of peroxisomal acylCo-A oxidase (ACOX1) and glyceronephosphate O-acyltransferase (GNPAT) was performed using cultured neural cells. In neuronal F3-Ngn1 cells, ACOX1 and GNPAT silencing up-regulated ceramide galactosyltransferase (UGT8) mRNA expression, and down-regulated UDP-glucose ceramide glucosyltransferase (UGCG). These results suggest that both impaired beta-oxidation of very long chain fatty acids and plasmalogen synthesis affect glycolipid metabolism in neuronal cells.