Wellcome Trust Centre For Mitochondrial Research

Nishani Jeyapalan – Euromit 2017 Abstract

Investigating mitochondrial trafficking within INGN2 neurons with large scale mitochondrial DNA deletions

Nishani Jeyapalan1, Oliver Russell1, Christopher Morris2, Doug Turnbull1 & Amy Reeve1

1Wellcome Trust Centre for Mitochondrial Research Medical School, Framlington Place, Newcastle University, Newcastle Upon Tyne, NE2 4HH
2Wolfson Building, Framlington Place, Newcastle University, Newcastle Upon Tyne, NE2 4HH

Introduction: Parkinson’s disease (PD) is a neurodegenerative disorder resulting in the loss of dopaminergic (DA) neurons from the substantia nigra pars compacta of the midbrain. Accumulation of large scale mitochondrial deletions contributes to mitochondrial dysfunction in PD; believed to be a contributor to DA neuronal loss in sporadic PD.

Aims: We observed the impact of mitochondrial dysfunction, caused by a large scale mitochondrial DNA deletion on mitochondrial trafficking in INGN2 neurons.

Methods: Human fibroblasts were converted into induced neuronal (iN) cells via a single transcription factor; Ngn2. INGN2 stem cells were differentiated into neurons, to observe differences in mitochondrial movement in INGN2 cell lines with lower (<10 %) and higher (<50 %) levels of mtDNA heteroplasmy. Cultured INGN2 neurons were loaded with TMRM prior to live cell imaging. Imaging of neurons was performed on a Nikon (Inverted confocal) A1r. Videos were obtained at 5 frames/second for 10 minutes for both high and lower heteroplasmy neurons.

Findings:  Analysis of mitochondrial movement was conducted on Imaris, where mitochondrial track length, mean speed, track duration, mitochondrial membrane potential and mitochondrial volume were measured and compared between both cell lines. Our studies demonstrated that the higher heteroplasmy INGN2 cell line exhibited faster axonal mitochondrial movement in comparison to the lower heteroplasmy INGN2 cell line. Mitochondria also moved over a shorter track duration which covered equal distances travelled to that of mitochondria with lower levels of heteroplasmy. High heteroplasmy INGN2 neurons also demonstrated a reduced mitochondrial membrane potential in comparison to cell line with lower levels of heteroplasmy.

Conclusion: We propose that mitochondrial trafficking within the high heteroplasmy INGN2 cell line, may exhibit a compensatory mechanism which transports mitochondria to high energy requiring regions within the neuron.