Metabolism of iron and iron oxide nanoparticles in glial cells

Iron is an essential metal for mammalian cells catalyzing redox reactions in various metabolic pathways. However, iron can also induce cellular damage due to increased formation of reactive oxygen species (ROS). Among the different brain cell types, oligodendrocytes produce and maintain the myelin sheaths around neuronal axons whereas brain astrocytes participate in a variety of different brain functions such as synaptic signal transduction, regulation of metal homeostasis and detoxification of xenobiotics. In brain, these cells may encounter iron oxide nanoparticles (Fe-NP), since Fe-NP are extensively investigated for biomedical applications. This thesis investigated the metabolism of iron and Fe-NP in glial cells. The oligodendroglial OLN-93 cells express the mRNAs of the protein transferrin, transferrin-receptor and divalent metal transporter 1 for iron uptake as well as the iron storage protein ferritin. The proliferation of these cells depended on the availability of extracellular iron and can be inhibited by iron chelators. Furthermore, OLN-93 cells accumulated substantial amounts of iron from low molecular weight iron salts and Fe-NP. The cell viability was not compromised despite of high intracellular iron concentrations. Moreover, exposure to Fe-NP hardly affected the metabolism of OLN-93 cells. Intracellularly, iron was mobilized from Fe-NP by OLN-93 cells as demonstrated by the increase in proliferation following iron restriction, by the upregulation of ferritin and by the inhibition of Fe-NP-dependent ROS formation by a cell-membrane-permeable iron chelator. Also primary astrocytes took up Fe-NP as shown by increased cellular iron contents and electron microscopy. Both OLN-93 cells and astrocytes accumulated iron from Fe-NP in comparable amounts, showed similar time- and concentration-dependencies of iron accumulation and stored iron in ferritin. These observations suggest that the uptake and the cellular fate of Fe-NP are similar in OLN-93 cells and astrocytes

Metabolismus von Eisen und Eisenoxid-Nanopartikeln in Gliazellen

Iron is an essential metal for mammalian cells catalyzing redox reactions in various metabolic pathways. However, iron can also induce cellular damage due to increased formation of reactive oxygen species (ROS). Among the different brain cell types, oligodendrocytes produce and maintain the myelin sheaths around neuronal axons whereas brain astrocytes participate in a variety of different brain functions such as synaptic signal transduction, regulation of metal homeostasis and detoxification of xenobiotics. In brain, these cells may encounter iron oxide nanoparticles (Fe-NP), since Fe-NP are extensively investigated for biomedical applications. This thesis investigated the metabolism of iron and Fe-NP in glial cells. The oligodendroglial OLN-93 cells express the mRNAs of the protein transferrin, transferrin-receptor and divalent metal transporter 1 for iron uptake as well as the iron storage protein ferritin. The proliferation of these cells depended on the availability of extracellular iron and can be inhibited by iron chelators. Furthermore, OLN-93 cells accumulated substantial amounts of iron from low molecular weight iron salts and Fe-NP. The cell viability was not compromised despite of high intracellular iron concentrations. Moreover, exposure to Fe-NP hardly affected the metabolism of OLN-93 cells. Intracellularly, iron was mobilized from Fe-NP by OLN-93 cells as demonstrated by the increase in proliferation following iron restriction, by the upregulation of ferritin and by the inhibition of Fe-NP-dependent ROS formation by a cell-membrane-permeable iron chelator. Also primary astrocytes took up Fe-NP as shown by increased cellular iron contents and electron microscopy. Both OLN-93 cells and astrocytes accumulated iron from Fe-NP in comparable amounts, showed similar time- and concentration-dependencies of iron accumulation and stored iron in ferritin. These observations suggest that the uptake and the cellular fate of Fe-NP are similar in OLN-93 cells and astrocytes

Glutamate dehydrogenase is essential to sustain neuronal oxidative energy metabolism during stimulation

The enzyme glutamate dehydrogenase (GDH; Glud1) catalyzes the (reversible) oxidative deamination of glutamate to α-ketoglutarate accompanied by a reduction of NAD+ to NADH. GDH connects amino acid, carbohydrate, neurotransmitter and oxidative energy metabolism. Glutamine is a neurotransmitter precursor used by neurons to sustain the pool of glutamate, but glutamine is also vividly oxidized for support of energy metabolism. This study investigates the role of GDH in neuronal metabolism by employing the Cns- Glud1-/- mouse, lacking GDH in the brain (GDH KO) and metabolic mapping using 13C-labelled glutamine and glucose. We observed a severely reduced oxidative glutamine metabolism during glucose deprivation in synaptosomes and cultured neurons not expressing GDH. In contrast, in the presence of glucose, glutamine metabolism was not affected by the lack of GDH expression. Respiration fuelled by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during an elevated energy demand, and it may reflect the large allosteric activation of GDH by ADP

PARK2 Mutation Causes Metabolic Disturbances and Impaired Survival of Human iPSC-Derived Neurons

The protein parkin, encoded by the PARK2 gene, is vital for mitochondrial homeostasis, and although it has been implicated in Parkinson's disease (PD), the disease mechanisms remain unclear. We have applied mass spectrometry-based proteomics to investigate the effects of parkin dysfunction on the mitochondrial proteome in human isogenic induced pluripotent stem cell-derived neurons with and without PARK2 knockout (KO). The proteomic analysis quantified nearly 60% of all mitochondrial proteins, 119 of which were dysregulated in neurons with PARK2 KO. The protein changes indicated disturbances in oxidative stress defense, mitochondrial respiration and morphology, cell cycle control, and cell viability. Structural and functional analyses revealed an increase in mitochondrial area and the presence of elongated mitochondria as well as impaired glycolysis and lactate-supported respiration, leading to an impaired cell survival in PARK2 KO neurons. This adds valuable insight into the effect of parkin dysfunction in human neurons and provides knowledge of disease-related pathways that can potentially be targeted for therapeutic intervention