Poster Z8 - Microglia Mitochondria Transfer and the Effects on Neuronal Development and Bioenergetics

Introduction: Mitochondria (mt) and their function are essential to the brain, especially neurons. Studies demonstrated that in healthy conditions, neurons receive mitochondria from a donor cell, microglia, as a way to increase ATP production, normalize calcium levels, and restore mitochondrial membrane potentials. However, the mechanism of transfer is unknown, nor if a cell-to-cell connection is required for mitochondrial uptake. No studies have yet determined the fundamental role of microglial mitochondria transfer in neuronal development and function, especially with respect to alterations in their bioenergetic profile. Our study aims to fill this gap by examining the alterations in the bioenergetic profiles of naïve neuron (N) cultures when treated with microglial (M) mitochondria compared to a neuron + microglia (NM) culture.

Materials and

Methods: To understand the cell-specific contribution of mitochondrial acceptance, alterations in bioenergetic profiles, and the mechanism associated with mitochondria transfer, we employed a human 3D in vitro model containing either neurons (DsRed2 mt), microglia (GFP mt), or neurons + microglia with fluorescent mitochondria. NM cultures were monitored via immunofluorescent imaging from seeding to determine when the transfer of mitochondria between the cell types occurs. To determine if a physical connection between the two cell types is required, freshly isolated mitochondria were dosed to naïve neuronal cultures. At 24 and 48 hours, we monitored the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) via the Seahorse XF Pro Analyzer. This was correlated with immunofluorescent images showing the visible acceptance of microglial mitochondria in neurons.

Results, Conclusions, and Discussions: In the NM cultures, we observed that mitochondria were transferred to neurons during development, around 3 weeks post-seeding. 24 hours post-treatment with microglial mitochondria, there is no visible acceptance by neurons. Its OCR and ECAR results confirm this, with high variability associated with this time point. At the 48-hour timepoint, there is visible microglial mitochondrial acceptance, with a significant difference in the number of intracellular mitochondria present in the treated versus untreated groups (p-value < 0.05) and no significant difference between the treated group and NM control. Comparing the trends in intracellular ECAR, we observed a significant decrease in the treated group compared to the untreated and NM control groups (p-value < 0.05). In addition, a similar trend can be observed for intracellular OCR, with a significant difference between all three conditions (p-value < 0.05).

Overall, NM cultures have higher ATP production compared to the treated and untreated cultures. This aligns with our observation of microglial mitochondria transfer to neurons in NM cultures 3 weeks post-seeding. While we see a significant difference in the amount of mitochondria between the treated and untreated conditions, there is no statistical difference between the untreated and NM control. This can be attributed to the fusing of neuronal and microglial mitochondria, which is displayed in immunofluorescent images of NM cultures. In both intracellular ECAR and OCR, the treated group had a significant decrease in ATP production, suggesting that mitochondria were accepted after 48 hours. However, this accepted mitochondrion is non-functional compared to the NM cultures, decreasing overall ATP production. This shows the critical role of the neuron-glial cell connection in the brain and its requirement for proper functionality. While the neurons are able to reuptake mitochondria from their external environment, it is not inherently beneficial to the neuron’s function.

Our findings underscore the critical role of microglia in neuronal development and the necessity of a physical connection between microglia and neurons for the beneficial transfer of mitochondria. These insights deepen our understanding of the brain's bioenergetics and provide a solid foundation for further research on the role, benefits, and mechanisms associated with mitochondrial transfer in the brain.

Acknowledgements (Optional): We thank the University of Cincinnati Liaudanskaya Start-Up Fund, Faculty Scholar Award, and Cincinnati Children's Bio-Imaging Analysis Facility.