RESUMO
Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-ß-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.
Assuntos
Cobre , Síndrome dos Cabelos Torcidos , Camundongos , Animais , ATPases Transportadoras de Cobre , Cobre/metabolismo , Plexo Corióideo/metabolismo , Síndrome dos Cabelos Torcidos/metabolismo , Encéfalo/metabolismoRESUMO
Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.
Assuntos
Aldeídos , Química Encefálica , Espectrometria de Massas em Tandem , Animais , Espectrometria de Massas em Tandem/métodos , Aldeídos/análise , Aldeídos/química , Camundongos , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Masculino , Modelos Lineares , Encéfalo/metabolismo , Limite de Detecção , Camundongos Endogâmicos C57BLRESUMO
Lipid peroxidation is a key component in the pathogenesis of numerous disease states, where the oxidative damage of lipids frequently leads to membrane dysfunction and subsequent cellular death. Glycerophosphoethanolamine (PE) is the second most abundant phospholipid found in cellular membranes and, when oxidized, has been identified as an executor of ferroptotic cell death. PE commonly exists in the plasmalogen form, where the presence of the vinyl ether bond and its enrichment in polyunsaturated fatty acids make it especially susceptible to oxidative degradation. This results in a multitude of oxidized products complicating identification and often requiring several analytical techniques for interpretation. In the present study, we outline an analytical approach for the structural characterization of intact oxidized products of arachidonate-containing diacyl and plasmalogen PE. Intact oxidized PE structures, including structural and positional isomers, were identified using complementary liquid chromatography techniques, drift tube ion mobility, and high-resolution tandem mass spectrometry. This work establishes a comprehensive method for the analysis of intact lipid peroxidation products and provides an important pathway to investigate how lipid peroxidation initially impacts glycerophospholipids and their role in redox biology.
Assuntos
Plasmalogênios , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Plasmalogênios/química , Plasmalogênios/metabolismo , Oxirredução , GlicerofosfolipídeosRESUMO
Changes in plasmalogen glycerophosphoethanolamine (PE-P) composition (structure and abundance) are a key indicator of altered lipid metabolism. Differential changes in the levels of PE-P have been reported in different disease states, including neurodegenerative diseases. Of particular interest, traumatic brain injury (TBI) has resulted in altered expression of glycerophospholipid profiles, including PE-P. To date, most analytical assays assessing PE-P have focused on general lipidomic workflows to evaluate the relative, semi-quantitative abundance of PE-P during disease progression. This approach provides a broad evaluation of PE-P, yet often lacks specificity and sensitivity for individual PE-P structures which is a necessity for robust quantitative data. The present study highlights the development of a targeted, quantitative method using a HILIC separation and selective reaction monitoring mass spectrometry for the confident identification and accurate quantitation of PE-P. Our innovative method incorporates both the sn-1 alkyl vinyl ether and sn-2 acyl chain as product ion transitions, for specific and sensitive quantitation of 100 PE-P structures. Our method also uniquely allowed for the unambiguous assignment and quantitation of di-unsaturated sn-1 PE-P structures, which to date have not been conclusively quantified. Application of this assay to a TBI mouse model resulted in distinct temporal profiles for plasma PE-P up to 28 days post injury. Plasma PE-P were significantly increased 24 h after induced TBI, followed by a gradual reduction to sham concentrations by day 28. Overall, we established a structure-specific, quantitative assay for identification and quantitation of a comprehensive set of PE-P structures with demonstrated relevance to brain injury.