Live imaging of mitochondrial dynamics in CNS dopaminergic neurons in vivo demonstrates early reversal of mitochondrial transport following MPP+ exposure

Abstract

Extensive convergent evidence collectively suggests that mitochondrial dysfunction is central to the pathogenesis of Parkinson’s disease (PD). Recently, changes in the dynamic properties of mitochondria have been increasingly implicated as a key proximate mechanism underlying neurodegeneration. However, studies have been limited by the lack of a model in which mitochondria can be imaged directly and dynamically in dopaminergic neurons of the intact vertebrate CNS. We generated transgenic zebrafish in which mitochondria of dopaminergic neurons are labeled with a fluorescent reporter, and optimized methods allowing direct intravital imaging of CNS dopaminergic axons and measurement of mitochondrial transport in vivo. The proportion of mitochondria undergoing axonal transport in dopaminergic neurons decreased overall during development between 2 days post-fertilization (dpf) and 5dpf, at which point the major period of growth and synaptogenesis of the relevant axonal projections is complete. Exposure to 0.5 – 1.0mM MPP+ between 4 – 5 dpf did not compromise zebrafish viability or cause detectable changes in the number or morphology of dopaminergic neurons, motor function or monoaminergic neurochemistry. However, 0.5mM MPP+ caused a 300% increase in retrograde mitochondrial transport and a 30% decrease in anterograde transport. In contrast, exposure to higher concentrations of MPP+ caused an overall reduction in mitochondrial transport. This is the first time mitochondrial transport has been observed directly in CNS dopaminergic neurons of a living vertebrate and quantified in a PD model in vivo. Our findings are compatible with a model in which damage at presynaptic dopaminergic terminals causes an early compensatory increase in retrograde transport of compromised mitochondria for degradation in the cell body. These data are important because manipulation of early pathogenic mechanisms might be a valid therapeutic approach to PD. The novel transgenic lines and methods we developed will be useful for future studies on mitochondrial dynamics in health and disease.

Mitochondria at the neuronal presynapse in health and disease

“…Indeed, anterograde axonal transport of mitochondria is reduced in mouse dopaminergic neurons deficient in respiratory chain enzymes 165 or exposed to the neurotoxin 1-methyl-4phenylpyridinium (MPP + ), which also increases retrograde transport 166 . In vivo imaging in zebrafish has also revealed substantially increased retrograde movement of mitochondria in dopaminergic neurons exposed to MPP+ 167 . These findings highlight the importance of appropriate trafficking of mitochondria in response to neuronal stress.…”

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

“…Because no degradation events were directly observed within the neuronal segments being monitored, the simple counting arguments strongly imply that mitochondria are being transported out of the view fields for degradation elsewhere (see section 7.4 ). There is compelling evidence from in vivo studies that damaged mitochondria are transported from neuronal processes towards the cell body for degradation (Dukes et al, 2016). Alternative mechanisms may also exist to eliminate mitochondria from neuronal processes.…”

The role of mitochondria in axon development and regeneration

“…Similar observations were obtained through analysis of axonal mitochondria in the intercostal nerves of mice engineered to express mitochondrially targeted fluorescent proteins (Misgeld et al, ). In vivo imaging of mitochondria in the axons of developing Zebrafish sensory or dopaminergic central nervous system neurons expressing a mitochondrially targeted fluorescent protein also revealed a large proportion of stalled mitochondria and a smaller population of bidirectionally transported mitochondria (Dukes et al, ). Observations in live motor neuron axons in Drosophila larvae expressing mitochondrially targeted fluorescent proteins similarly showed a large proportion of stalled mitochondria (Louie et al, ).…”