<i>5XFAD</i> MOUSE MODEL: INVESTIGATING BRAIN IRON IMBALANCE AND MITOCHONDRIAL FEATURES’ ALTERATION

In the brain iron supports neuronal activity and neurotransmitter synthesis. The primary site of iron utilization is mitochondria where it is required for the formation of cofactors essential to redox reactions and energy metabolism, such as ATP production via the electron transport chain (OXPHOS). We previously showed that iron dyshomeostasis occurs with aging, and evidence links disrupted iron balance to mitochondrial dysfunction, energy loss and neuronal death in neurodegenerative diseases like Alzheimer’s disease (AD). The role of brain iron-driven mitochondrial impairment in AD remains unclear. Here, we examined iron homeostasis during the pre-symptomatic phase in 5xFAD mice, carrying five familial human AD mutations. At 2 months, 5xFAD mice showed marked brain iron accumulation in subcortical and striatal areas as revealed by Prussian blue Perl’s staining along with early intracellular amyloid-beta deposition. Due to mitochondria’s key role in iron trafficking and utilization, enriched mitochondrial fractions isolated from 5xFAD brains showed increased expression of mitochondrial number verified by TOM20, mitochondrial aconitase and OXPHOS complexes. Interestingly, we found a significant increase of mitochondrial ferritin, the iron stock within mitochondria, indicating altered iron handling. Moreover, the increased mitochondrial number is not due to de novo biogenesis, as shown by unchanged expression of Cytochrome b, NADH dehydrogenase 1, and PGC-1α; instead, it results from mitochondrial accumulation. Indeed, we found a marked reduction in markers of autophagy and of mitophagy, indicating compromised autophagy in 5xFAD mice and sustaining the previous results of mitochondria accumulation. Furthermore, accumulated mitochondria showed reduced ATP production and respiration while oxidative stress markers were increased. These findings reveal early disruptions in brain iron metabolism and mitochondrial turnover, highlighting mitochondrial iron as a potential target for Alzheimer’s disease. This work is supported by MIUR project “Dipartimenti di Eccellenza 2023-2027” to Department of Neuroscience “RitaLevi Montalcini”, Fondazione CRT “Richieste Ordinarie 2021”, UNITO and UNITA grants for Internationalization 2022 to Serena Stanga.