# MitoTracker transfers from astrocytes to neurons independently of mitochondria

## TL;DR

This study shows that MitoTracker dye can transfer from astrocytes to neurons without actual mitochondria being transferred, which is important for interpreting previous experiments.

## Contribution

The study demonstrates that MitoTracker dye transfer between astrocytes and neurons is independent of mitochondrial transfer and does not require cell contact.

## Key findings

- MitoTracker dye transfers to neurons from astrocytes and conditioned media independently of mitochondrial transfer.
- Genetically encoded GFP labeling did not show mitochondrial transfer from astrocytes to neurons.
- MitoTracker transfer does not equate to actual mitochondrial transfer.

## Abstract

The neuroprotective transfer of mitochondria from astrocytes to neurons has been primarily investigated by labeling astrocytic mitochondria with the dye MitoTracker. Here, we labeled astrocytic mitochondria with both a genetically encoded fluorophore (GFP) and MitoTracker dye and then imaged neurons immediately after co-culture with astrocytes or astrocyte-conditioned media (ACM). We report that MitoTracker transfers to neurons from both astrocytes and ACM, independently of mitochondrial transfer. Our observations provide an essential caveat to the use of this reagent and suggest that the investigation of astrocyte-neuron mitochondrial transfer, and other systems in which contact-independent transfer has been reported, requires the use of alternative labeling techniques.

•We labeled astrocyte mitochondria with GFP and MitoTracker before co-culture•We did not detect mitochondrial transfer from astrocytes to neurons•MitoTracker rapidly transferred from astrocytes and conditioned media to neurons•MitoTracker transfer does not equate to mitochondrial transfer

We labeled astrocyte mitochondria with GFP and MitoTracker before co-culture

We did not detect mitochondrial transfer from astrocytes to neurons

MitoTracker rapidly transferred from astrocytes and conditioned media to neurons

MitoTracker transfer does not equate to mitochondrial transfer

The phenomenon of intercellular mitochondrial transfer (IMT) from astrocytes to neurons is primarily supported by experiments using the dye MitoTracker to label astrocytic mitochondria. It was recently shown in other cell types that MitoTracker dye can transfer from donor to acceptor cells independently of mitochondrial transfer. However, MitoTracker dye transfer in these cells was dependent on cell-cell contact, while astrocyte-to-neuron IMT is reportedly contact independent. It remained unclear whether MitoTracker could similarly transfer between these cell types, independently of IMT. We aimed to clarify this by dual labeling astrocytic mitochondria with genetically encoded GFP and the MitoTracker dye. This enables direct comparison of dye versus mitochondrial transfer to neurons.

The mitochondrial dye MitoTracker is commonly used to investigate intercellular mitochondrial transfer (IMT), particularly between astrocytes and neurons. Hole et al. compare MitoTracker with a genetically encoded mitochondrial fluorophore and demonstrate that MitoTracker can transfer from astrocytes to neurons in the absence of mitochondrial transfer, without requiring cell contact.