This 2016 master's thesis analyzed the slow turnover of stationary mitochondria in axonal arbors of cerebellar granule neurons. The author found that about 84% of mitochondria are stationary, but undergo positional changes within hours. Mitochondrial turnover rate was not significantly different between young and old cultured cerebellar granule neurons, and did not depend on branch length. This study provides insight into the systems that regulate mitochondrial distribution and quality in neuronal axons.

1. 2016 Master thesis
Analysis of slow mitochondrial turnover in axonal arbors of cerebellar
granule neurons
小脳顆粒神経細胞の軸索におけるミトコンドリアの
遅い入れ替わりについての解析
September 2016
AL AMIN
ID: 14798018
Laboratory for the Regulation of Neuronal Morphology,
Department of Human and Artificial Intelligent Systems,
Graduate School of Engineering, University of Fukui

2. Abstract
Proper distribution of mitochondria within axons and at synapses is critical for
neuronal function. Mitochondria move bidirectional, pause briefly, and move again,
frequently changing direction. It has been reported that most of mitochondria in axon are
in the stationary state by anchoring to the microtubules. However, mechanisms by which
anchoring/displacement of stationary mitochondria remain largely unknown. In this study,
I genetically labeled mitochondria and performed time lapse imaging of mitochondria in
axons of primary cultured cerebellar granule neurons. I found that about 84%
mitochondria are present as stationary state. Importantly, I found that they undergo
turnover and most of mitochondria change their position within a few hours.
I also quantified the turnover of mitochondria in between young (5 days in vitro,
DIV) and old (10 DIV) CGNs and found that there is no significant difference.
Furthermore, I found that turnover rate of stationary mitochondria is not dependent of the
branch length. Together, I found the slow turnover of stationary mitochondria in axon,
which is not affected by neuronal maturation and branch-dependent regulation at least in
cultured CGNs. I think my present study will be valuable to understand the systems that
coordinately regulate mitochondrial quality and distribution.

3. Table of contents
1. Introduction and Background
1-1. Over view ..................................................................................1
1-2. Nerve cells ..................................................................................1
1-3. Cerebellar granule cells ..................................................................................2
1-4. Microtubules ..................................................................................3
1-5. Regulation of axonal mitochondria ...................................................................4
2. Experimental Methods
2-1. Cerebellar granule cell culture ..................................................................................3
2-2. Gene transfer by the calcium phosphate method ...................................................7
2-3. Time-lapse observation using a fluorescent microscope ....................................9
2-4. Image Analysis by using imageJ software ...................................................9
2-5. Composition of the reagents ..................................................................................10
2-6. Flowchart of the experiments ..................................................................................13
3. Results
3-1. Quantification of stational vs. motile mitochondria in CGN axons .....................14
3-2. Detection of turnover of stationary mitochondria in CGN axons .....................16
3-3. Comparison of turnover of mitochondria in young vs. old CGN axons .....................18
3-4. Analysis of region dependent difference of stationary mitochondria
in CGN axonal arbors ..................................................................................20
3-5. Analysis of region dependent difference in the turnover of stationary
mitochondria in CGN axons ..................................................................................23
4. Discussions ..................................................................................25
5. Acknowledgments ..................................................................................26
6. References ..................................................................................27