molecular and cellular insights into mitochondrial contributions to neuronal autophagy

458 Pages·2016·14.54 MB·English

by  Yea Seul Shin

MOLECULAR AND CELLULAR
INSIGHTS INTO MITOCHONDRIAL
CONTRIBUTIONS TO
NEURONAL AUTOPHAGY:
LINKS TO ENERGETICS AND
MITOPHAGY
By
Yea Seul Shin
Submitted in total fulfilment of the requirements of the degree of
DOCTOR OF PHILOSOPHY
Florey Institute of Neuroscience and Mental Health
Department of Anatomy and Neuroscience
Faculty of Medicine, Dentistry and Health Sciences
The University of Melbourne
February 2016

ABSTRACT
Neurones are essential for brain homeostasis and as highly metabolic cells rely on
mitochondrial oxidative phosphorylation (OXPHOS) for energy. The integrity and
functionality of mitochondria are critical for neuronal survival, and the involvement of
dysfunctional mitochondria is recognized as a common theme amongst various
neuropathologies. New evidence has suggested that the inappropriate clearance of
dysfunctional mitochondria via autophagy (termed mitophagy) determines the
pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Mechanistic
studies of mitophagy have been undertaken using mammalian cell lines, but this research
lacks relevance to neuropathologies. This thesis investigates triggers of
autophagy/mitophagy in primary neurones, and specifically if disruption of mitochondrial
bioenergetics triggers neuronal autophagy, and mitophagy in particular.
Cultures of primary cerebellar granule cells (CGCs) were utilized and inhibitors of
the OXPHOS complexes (rotenone, 3-Nitropropionic acid, antimycin A, potassium
cyanide and oligomycin targeting complex I-V, respectively), were employed to induce
bioenergetic dysfunction of mitochondria. Initial investigations using MTT cell viability
assay, phase contrast microscopy and cellular membrane permeabilization detected by
propidium iodide staining, determined appropriate concentrations of OXPHOS inhibitors
which induced effective mitochondrial damage producing slow neuronal degeneration.
From this baseline adverse effects of OXPHOS inhibitors on mitochondrial bioenergetics
were documented by monitoring reductions in cellular ATP level, mitochondrial
membrane potential (ΔΨ ) and oxygen consumption rate (OCR) of CGCs. ΔΨ was
m m
rapidly dissipated in CGCs exposed to the inhibitors of complexes I, III and IV (rotenone,
antimycin A and potassium cyanide, respectively), whilst the inhibitor of complex II, 3-
Nitropropionic acid, produced a much slower reduction of ΔΨ . Employing Seahorse
m
XF24 technology allowed an incisive readout of mitochondrial functional changes where
significant bioenergetic impairment was observed subsequent to inhibition of complexes I
and II, which are core components of energy metabolism regulating the redox balance
(NAD+/NADH levels) and TCA cycle.
Abstract
Existent evidence indicates depolarization of ΔΨ triggers mitophagy in
m
mammalian cell lines, however CGCs display contrasting results where ΔΨ
m
depolarization via complex III and IV inhibition was insufficient to elicit mitophagy
despite the inducer of mitophagy, PINK1, being mobilized to mitochondria. In contrast,
inhibition of complexes I and II induced mitophagy, as indicated by PINK1 mobilization
and disappearance of the pH-sensitive fluorescence mitophagy reporter, mt-Rosella.
Western immunoblotting of the general autophagy marker, LC3, and monitoring of acidic
vesicles with monodansylcadaverine revealed activation of autophagic flux in CGCs
exposed to inhibitors of complexes I-IV, indicating general autophagy in response to
bioenergetic impairment irrespective of mitophagy induction.

Results presented herein reveal the complexity of neuronal mitophagy and that
ΔΨ may not be a necessary trigger of neuronal mitophagy. Thus inhibition of individual
m
respiratory complexes, and notably complexes I and II, may underlie the triggering of
mitophagy in primary neurones, where different mechanisms induce mitophagy in
neurones compared to immortalized cell lines. This difference may be due to the unique
bioenergetic dependence of neurones. Understanding the mechanisms of mitophagy and
autophagy in primary neurones provides valuable insights into therapeutic approaches for
neurodegenerative diseases.
Abstract
Declaration

This is to certify that:

(i) the thesis comprises only my original work towards the PhD except where indicated,

(ii) due acknowledgement has been made in the text to all other material used,

(iii) the thesis is less than 100,000 words in length, exclusive of tables, figure legends,
bibliography and appendices.

Yea Seul Shin
Declaration
Publications
During the course of my studies the following manuscripts have been published.
Publication which is directly related to the work described in this thesis is denoted by *.
1. Sheean RK, Lau CL, Shin YS, O'Shea RD, Beart PM. Links between l-glutamate
transporters, Na(+)/K(+)-ATPase and cytoskeleton in astrocytes: evidence
following inhibition with rottlerin. Neuroscience. 254:335-46 (2013).
*2. Higgins GC, Beart PM, Shin YS, Chen MJ, Cheung NS, Nagley P. Oxidative
stress: emerging mitochondrial and cellular themes and variations in neuronal
injury. J Alzheimers Dis.;20 Suppl 2:S453-73 (2010) Review.
3. Diwakarla S, Mercer LD, Kardashsyan L, Chu PW, Shin YS, Lau CL, Hughes
ML, Nagley P, Beart PM. GABAergic striatal neurons exhibit caspase-
independent, mitochondrially mediated programmed cell death. J Neurochem.109
Suppl 1:198-206 (2009).
Abstracts
1. Shin YS, Ryall JG, Britto JM, Higgins GC, Beart PM and Nagley P (2014).
Mitochondrial contributions to neuronal autophagy: links to energetics and
mitophagy? FAOBMB, Hong Kong.
2. Shin YS, Ryall JG, Britto JM, Higgins GC, Nagley P and Beart PM (2014).
Mitochondrial contributions to neuronal autophagy: Links to energetics and
mitophagy? ANS, Adelaide. Proc Aust Neurosci Soc. 25.
Publications & Abstracts
3. Shin YS, Higgins GC, Nagley P and Beart PM (2013). Mitochondrial
contributions to neuronal programmed cell death: evidence for an interface of
autophagy with mitochondrial energetics. ANS, Melbourne. Proc Aust Neurosci
Soc. 24

4. Shin YS, Mercer LD, Higgins GC, Nagley P and Beart PM (2012).
Mitochondrial contributions to neuronal programmed cell death: evidence for
involvement of autophagy. APSN/JSN; Kobe, Japan. J Neurochemistry, 123
(Suppl. 1), 48–88.

5. Shin YS, Higgins GC, Nagley P and Beart PM (2011). Mitochondrial
contributions to neuronal injury of cerebellar granule cells: evidence for
recruitment of autophagy. ANS, Auckland. Proc Aust Neurosci Soc. 22

6. Shin YS, Chu PWY, Mercer LD, Higgins GC, O’Shea RD, Nagley P and Beart
PM (2009). Mitochondrial injury and programmed cell death including apoptosis,
autophagy and endoplasmic reticulum stress in neuronal injury. 22nd Biennial
Meeting of the ISN/APSN, Busan, South Korea. J. Neurochem. 110 (Suppl. 2),
24–66

7. Shin YS, Chu PWY, Nagley P and Beart PM (2009). Programmed cell death in
cerebellar granule cells induced by inhibition of mitochondrial respiratory chain
complexes. ANS, Canberra. Proc Aust Neurosci Soc. 20

8. Mercer LD, Diwakarla S, Chen B, Kardashyan L, Chu P, Shin YS, Lau CL, Lim
M, Nagley P and Beart PM (2009). Phenotyping neuronal injury: Insult
dependent crosstalk between caspase dependent/-independent cell death pathways.
ANS, Canberra. Proc Aust Neurosci Soc. 20
Publications & Abstracts

Acknowledgements

The completion of this thesis would not have been achieved without the support and
contribution of a special group of people to whom I am indebted.
First, I would like to express tremendous appreciation and thanks to my primary
supervisor, Professor Phil Beart, who has supervised me with great knowledge and
insight - with patience and support. Finishing this incredible PhD journey would have
been impossible without his ceaseless encouragement. Phil, I do not think there are
enough words to describe how thankful I am to have been your student. It was such a
privilege to closely observe and learn how you think and write as a scientist and how you
value and mentor other upcoming research fellows. Thank-you so much for teaching me
how important it is to maintain a good life and work balance. I am grateful that you were
always available for me, even during the busiest times of the year (although there were
mysterious times when you became ‘invisible’ when you were on your way to the gym!).
Thank-you so much, this page is too small to hold all my gratitude towards you. I really
could not have asked for a better supervisor. I would also like to thank and acknowledge
Professor Phillip Nagley for sharing his exceptional insight on mitochondria and for all
his mentoring and guidance. Phillip, I truly appreciated and respected every single piece
of advice that you have given me. Your advice shaped my attitude towards the
completion of my PhD and gave me courage to write one more word. Your way of
teaching and patient mentoring motivated me greatly and I took a brave step into teaching
following your lead. Thank-you for showing me how to respond and battle through life’s
hardest moments. You were a great encourager and you still are.
I am indebted to all of the members of the Beart laboratory. Thank you for making the lab
a great place to be. I wish to thank Senior Research Officer, Linda Mercer, who has been
completely supportive during my journey battling with injuries and getting back to work.
Thank-you for being my Melbourne-mum, for giving me lots of warm hugs and
encouragement along this long journey. All of your gifts are my best treasures! I would
also like to thank Dr. Linda Lau. Thank-you so much for all your advice in the lab and for
being a good friend. I respect your great technical knowledge in our research area and I
also admire your gentleness and compassion towards people around you. A big thanks
goes to Drs. Percy Chu and Shanti Diwakarla for teaching me all the cell culturing
techniques when I first started as a PhD student. To Bec, Nicky, Malene, Cecilia, Sam,
Acknowledgements
Tine and Natasha, thank-you for lightening up the lab with lots of laughter and countless
coffee times!
I am grateful to my mentor, Dr Colleen Thomas. I will never forget the day when you
knocked on the door of my house in New Zealand during the Easter break in 2011. You
flew all the way from Melbourne to Auckland to pay me a visit while I was taking a sick-
leave! You have been a great encourager, listener and a good friend. Thank-you so much!
And to my other mentor Dr Gavin Higgins, thank-you for being a good mentor since the
beginning of my PhD candidature. Your invaluable technical advice in planning
experiments has helped me greatly. I hope to become a mentor like you, to other students
I may have the privilege to work with.
My sincere thanks also goes to Dr. Jo Britto for making my live cell imaging studies
possible. Thank-you for making time for me during the busiest times of the year and for
all your technical advice. Dr James Ryall allowed me to perform experiments using the
Seahorse XF24 analyzer and he gave insightful advice on the experiment results. Also, a
big thanks goes to Dr Janetta Culvenor for providing PINK1 antibodies. Without your
precious support, it would not have been possible to conduct this research.
My special thanks are also extended to Professor Rod Devenish for sharing his deep
knowledge on autophagy and for proof reading this section of my thesis, and to Drs.
Carlos Rosado and Dalibor Mijaljica for their valuable advice on the genetic engineering
of the mt-Rosella constructs. I would like to thank my Higher Degrees by Research
committee members, Dr. Holly Cate and Dr. Bradley Turner, for giving me valuable
advice each year during my annual PhD candidature meetings. Clinician and Florey
researcher Professor John Drago also generously gave me numerous neurological
consultations to help me manage the health issues that arose during my PhD candidature.
I take this opportunity to express gratitude to all of my wonderful friends. San Young,
you’re a true blessing, one dear friend whom I treasure so much. Thank-you for bringing
my sanity back whenever I went a little bit crazy. Dear Chris, thank-you for believing in
me and always being there for me. My dear sister, Yun Su, you know I love you! Sonic,
Sung Bae, Soo Hyun, Daniel and all the church family, you’ve seen my journey through
PhD from the beginning, I could not have made it without you guys. And thank-you
Ronny for your kindness for providing me with an accommodation during the hardest
time of my candidature.
Acknowledgements