Background

Mitochondria are double-membraned organelles present in essentially all the cells of every complex organism. Their main function is to supply the majority of cellular energy as ATP, but they also play roles in apoptosis, buffering intracellular Ca²⁺, reactive oxygen species production, and regulation of membrane potential (Neupert and Herrmann, 2007; Hamanaka and Chandel, 2010; Shutt and McBride, 2013).

These organelles, often depicted as single “bean-like” structures, are in reality components of a dynamic cytoplasmic network. They can undergo major morphological changes regulated by dynamic processes of membrane fusion and fission, a process believed to be involved in the elimination of dysfunctional organelles through a process called mitophagy. The mitochondrial network can also increase as a response to high cellular energy demand (Sheng, 2017; Devine and Kittler, 2018). The morphology of the mitochondrial network can be altered in response to different stressors, and there is a wide range of possible morphologies which are cell-type, or even cell-compartment dependent (Picard et al., 2013). The localization of mitochondria in the dendrites and axons of neurons, in particular, play a crucial role as they provide, in these specific cells, the energy necessary for synaptic transmission (Chang and Reynolds, 2006; Misgeld and Schwarz, 2017).

Mitochondria localization and dynamics in one particular group of cells, the dopaminergic neurons in the substantia nigra, have been extensively studied during the last decades. These neurons’ projections reach the striatum and their loss causes a depletion of striatal dopamine which is the cause of the classical motor symptoms of Parkinson’s disease (PD) (Dauer and Przedborski, 2003; Braak et al., 2004).

The involvement of mitochondrial dysfunctions in these neurons has been investigated since the 80’s (Kopin and Markey, 1988), but the study of mitochondrial dynamics attracted particular interest especially since the discovery that genes mutated in monogenic forms of Parkinson’s disease (in particular Parkin and PINK1) have an essential role in mitochondrial fission/fusion, mitochondrial transport and mitophagy (Koh and Chung, 2010; Narendra and Youle, 2011).

We recently investigated the consequences of the loss of Parkin in a mouse model of PD in which degeneration of dopaminergic neurons was caused by mtDNA depletion and mitochondrial dysfunction (Pinto et al., 2018). In this context, we also found that lack of Parkin affected mitochondrial morphology in dopaminergic axons.

A challenge in studying the mitochondria morphology in vivo is the clear visualization of the organelles and in discerning the ones present in the axons. To study mitochondrial morphology in dopaminergic neurons, we used immunofluorescence microscopy on mice specifically expressing eYFP in the mitochondria of dopaminergic neurons, as well as computerized image analysis software to study mitochondrial number, density and size, as a measure of mitochondrial health (Pinto et al., 2018).