Ncoa4-mediated Ferritinophagy in Relation to Neuronal Iron Usage During Mouse Brain Development
Iron in the brain is extremely important across the lifespan, but specifically in the developing brain due to its requirement for important processes including myelination, energy metabolism, neurotransmitter synthesis, and the regulation of gene expression. Ferritin protein complexes in the cytosol are responsible for cellular iron storage in the brain. As brain iron utilization changes for processes such as growth and differentiation, ferritin provides a ready reservoir of iron. The iron-binding ferritin protein is composed of two subunits–heavy and light. Within non-brain cells (e.g., red blood cells), the nuclear receptor coactivator 4 (NCOA4) protein assists in the distribution of intracellular iron and has been identified as a selective autophagic cargo receptor that initiates iron release from ferritin, termed ferritinophagy. Whether NCOA4 is expressed in the developing brain and important for iron-dependent brain development is unknown. This study aimed to determine how brain gene expression for proteins involved in ferritinophagy changes across in-vivo normal brain development. Dissections were conducted at three time points (postnatal day 0, 7, and 14) to obtain the cortex, cerebellum, and hippocampus from iron-sufficient mice (n=6 mice per age). Total RNA extraction and purification was conducted and the purified RNA was reverse transcribed to synthesize cDNA for quantitative polymerase chain reaction (qPCR). qPCR was used to quantify mRNA levels for Ncoa4, ferritin light chain (Ftl1), ferritin heavy chain (Fth1), and several potential reference genes. Data analysis to calculate relative mRNA levels was performed and the results were analyzed using a two-way ANOVA. No statistically significant differences were found in Ncoa4 or Ftl1 expression across time points or brain regions. Fth1 mRNA expression increased between P7 and P14 (p=0.0073) in the cerebellum and between P0 and P14 (p=0.0138) in the cortex. These Fth1 results are consistent with its known role in iron utilization and the increase in iron-demanding processes during this stage of brain development. We now know that Ncoa4 is expressed throughout the developing brain; however, it is not developmentally regulated at the mRNA level. This is consistent with Ncoa4’s role in ferritinophagy in other tissues, where Ncoa4 is regulated by iron at the level of protein stability. Next steps will include determining iron and developmental regulation of Ncoa4, Ftl1, and Fth1 protein abundance in the brain.