Live Imaging Mitochondrial Transport in Neurons

Abstract

Mitochondria are among a cell's most vital organelles. They not only produce the majority of the cell's ATP but also play a key role in Ca²⁺ buffering and apoptotic signaling. While proper allocation of mitochondria is critical to all cells, it is particularly important for the highly polarized neurons. Because mitochondria are mainly synthesized in the soma, they must be transported long distances to be distributed to the far-flung reaches of the neuron-up to 1 m in the case of some human motor neurons. Furthermore, damaged mitochondria can be detrimental to neuronal health, causing oxidative stress and even cell death, therefore the retrograde transport of damaged mitochondria back to the soma for proper disposal, as well as the anterograde transport of fresh mitochondria from the soma to repair damage, are equally critical. Intriguingly, errors in mitochondrial transport have been increasingly implicated in neurological disorders. Here, we describe how to investigate mitochondrial transport in three complementary neuronal systems: cultured induced pluripotent stem cell-derived neurons, cultured rat hippocampal and cortical neurons, and Drosophila larval neurons in vivo. These models allow us to uncover the molecular and cellular mechanisms underlying transport issues that may occur under physiological or pathological conditions.

Keywords: Axonal transport; Drosophila; Embryonic neurons; Larva; Live imaging; Microtubules; Mitochondrial transport; Neurodegenerative disease; iPSCs.

Fig. 1

Mitochondrial movement in an iPSC-derived neuronal axon. This representative iPSC-derived neuronal axon from a healthy subject was transfected with mito-dsRed. (A) The first frame of the time-lapse image series is shown above of a kymograph generated from the movie. The x-axis corresponds to mitochondrial position and the y-axis corresponds to time (progressing from top to bottom). Vertical white lines represent stationary mitochondria and diagonal lines are moving mitochondria. Scale bar = 10 μm. (B) A hand-drawn depiction of the kymograph shown in (A). Black lines label mitochondria when they are static, and blue or red lines label mitochondria when moving anterograde or retrograde, respectively

Fig. 2

Mitochondrial movement in a rat cortical neuronal axon. This representative cortical neuronal axon is transfected with mito-dsRed at 8 days in vitro. The kymograph is generated as in Fig. 1A.Scale bar = 10 μm

Fig. 3

Mitochondrial movement in a fly larval axon. Mito-GFP is expressed in a CCAP-expressing neuropeptidergic axon of a third instar larva. The representative segmental nerve passes hemisegment A3, with the VNC to the left. The kymograph is generated as in Fig. 1A. Scale bar = 10 μm