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Swimming exercise reduces native ⍺-synuclein protein species in a transgenic C. elegans model of Parkinson’s disease and confers downstream protective effects independent of food restriction…

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Swimming exercise reduces native ⍺-synuclein protein species in a transgenic C. elegans model of Parkinson’s disease and confers downstream protective effects independent of food restriction…

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Swimming exercise reduces native ⍺-synuclein protein species in a
transgenic C. elegans model of Parkinson’s disease and confers
downstream protective effects independent of food restriction:

Exploring Exercise as a Medicine for Parkinson’s disease.

by
Minna Y. Schmidt

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY IN BIOLOGY OF AGING

December 2021

Copyright 2021 Minna Y. Schmidt
ii

“Running water does not rot, and the door hinge does not corrode with rust because it is in
constant motion. It's the same with humans. If a person does not move, he does not set his energy
in motion and remains inert.”

— Sima Qian (145/135 BCE-86 BCE, court scribe, astrologer, and historian under Emperor Wu
Ti of the Han Dynasty)

iii

In memory of “Dadya Borya,” Dr. Boris Tsarenkov
(June 19, 1930-May 16, 2020)

iv
Acknowledgements
I would like to express my first point of gratitude to all of the wonderful teachers and
mentors I have had throughout my educational life. I would like to first thank my teachers at
Brandeis Hillel Day School — those who taught me personally, and those who I saw daily and
who played such an important role in shaping my environment. I would like to especially thank
Mrs. Calhoun, Mr. David Jeffries, and Mr. Chaim Heller. Throughout all of these years, I vividly
remember your warmth, love towards education, and great moral courage. You formed the
foundation upon which I have continued to build my educational journey, and I feel incredibly
thankful and inspired by you.
I would also like to thank my teachers at Lowell High School, in particular Dr. da Rosa,
Mr. Schwartz, Mr. Granucci, Mrs. Dramen, Mr. Axe, and Mr. Cohen. Your passion for your
subject matter, and desire to help your students achieve their very best potential, has served as
the key to our success.
I would like to extend my third point of gratitude to Dr. Jason Pontrello, my mentor and
PI at Brandeis University. While learning organic chemistry, and then applying my knowledge in
the laboratory, I first came to experience the freedom of thought in science, as well as my path as
a scientist and as an individual. Your seemingly never-ending patience, Dr. Pontrello, was
instrumental in fostering a love for, and understanding of chemistry in all of your students, and I
am very grateful to you.
I would also like to thank Dr. Judy Herzfeld. Judy, I have always cherished how you
never dismissed the ideas or thoughts of your students. You strived to understand and see things
as we saw them. In this way, we grew to understand ourselves, and chemistry better. Thank you.
v
Finally, I would like to address the great mentorship I have received at The Buck
Institute. I would like to first thank my committee members, Dr. Lisa Ellerby, Dr. Sean Curran,
and Dr. Simon Melov. Thank you for your thoughtful insight into this work. Thank you as well
for leading by example. From the initiation of my academic pursuits in this program, you have
been the voices in my head. I greatly appreciate your mentorship.
Thank you also to all past and present Andersen and Lithgow lab members. Manish, you
worked closely with me and fostered my knowledge and appreciation for the C. elegans worm. I
have really grown to love these little guys, and honestly, would not like to imagine my future
work without them! Your effortless love and curiosity towards science is deeply inspiring. Thank
you as well to Shankar. Shankar, I appreciate very much how you welcomed me to the Andersen
laboratory and worked closely with me in the beginning. You always encouraged me in my
work, patiently answered my questions, and supported me if I chose to pursue a unique approach.
I am also greatly thankful to Josue. Josue, you are so passionate about your work and it is an
honor to speak with you. You also greatly care about the problems or interests other scientists
may have. I do not know many people brave enough to listen to an unremitting spiel about
hypoxia from someone they barely know. I greatly cherish this conversation. To Harshi, your
dedication to science is incredible. You seem to have an unlimited amount of time to devote to
anyone who needs your help. You are a great inspiration to me. To Chaska, your great strength
of character, energy, and passion for your work, as well as anything else you pursue, is
incredibly inspiring. I am greatly thankful for your help and for your friendship. To Anand, I
owe great, great thanks. Anand, your precision, calm and welcoming personality, and great sense
of responsibility for the laboratory and each individual person has not only inspired me in my
vi
scientific work, but has time and time again, reminded me of the kindness and humanity we all
possess.
And finally, I would like to express how deeply appreciative I am of my mentors and
PI’s, Drs. Julie Andersen and Gordon Lithgow. Gordon, you instill such a great sense of
camaraderie, emphasizing both scientific and personal collaborations. You provide support in all
its forms, and you joyfully welcome the pursuit of all new ideas. Thank you so much for
listening (especially to the 18 different ideas), asking questions, and always being so warm and
welcoming. Julie, you have given me all of the mental and physical support I could ask for.
About eight years ago, it became my goal to study Parkinson’s, and my dream to be able to
answer some of its questions. After meeting you a few years later, I understood that this would
be possible exactly in your laboratory and with your guidance. I could not ask for anything
better. Thank you so much.
I would like to express my final point of thanks to all of my friends and family. About
thirty-two years ago, my parents sought, and were granted, asylum in the United States of
America. In their native country, they lived under difficult and sometimes dangerous
circumstances. When they arrived here, they had very little. Through the care of the American
community that aided their immigration, they were given all possible means to exist and to
become self-reliant. All of their lives, they strived to live in freedom, and they never relented to
raise me with these same ideals. Of all the values, and possible pursuits in life, they encouraged
me most to achieve the best education that I could. Having known about the possibility of losing
everything, they taught me that my only true and dear possession should be my knowledge. This
work is as much my effort as it is theirs. Thank you, mom and dad.
vii
In my last remark, I would like to express my gratitude to my dear uncle, Dr. Boris
Tsarenkov. Dadya Borya, dad always says how you have always been his “greatest teacher,” and
it is no question that you are mine as well. No matter what you would ask me, and no matter
what I would answer, you always looked at me in such a way that I only grew in confidence. If
we disagreed, you never dismissed my opinion. It is no wonder that you were so loved by all of
your students. You were always patient, calm, and confident, and you took on every problem
with limitless interest. You have always inspired me, and continue to inspire me and our family.
Although we had hoped to celebrate the completion of this work together in person, I can tell you
that we were already celebrating when we spoke two weeks before you left. At that time, I told
you how I came to a very clear understanding of something, and here, as you can see, we have
begun the ground work. I am very excited about the path we have taken, and I can certainly say,
it wouldn’t have been possible without you. I remember that when I told you about our idea, you
sighed with relief and happiness. I will continue to update you as we go along.

viii
TABLE OF CONTENTS
Acknowledgements.........................................................................................................................iv
List of Figures ................................................................................................................................xi
List of Abbreviations....................................................................................................................xiii
Abstract .......................................................................................................................................xvii
Preamble……………………………………………………………………………………….xviii
Introduction .....................................................................................................................................1

Chapter 1: Effects of a short bout of swimming exercise on native human ⍺-synuclein protein
species*…………………………………………………………………………………………..22

I. Effects of exercise and food restriction upon native human ⍺-synuclein
Protein species………………...…………………………………………………………23

II. Analysis of predominant ⍺-synuclein species present after exercise
and food restriction treatments…………………………………………………………...25

III. Observing ⍺-synuclein protein species in whole tissue using confocal
microscopy…………………………………………………………………………….....28

IV. Summary: How a short treatment of exercise and food restriction in N5901 worms
effects changes in native human ⍺-synuclein protein species…………………………...37

Chapter 2: Observing effects of longer versus shorter exercise and food restriction treatment
times……………………………………………………………………………………………...39

I. Observing changes in native ⍺-synuclein species within 15 minutes of swimming
exercise…………………………………………………………………………………..40

II. Timed experiment observing NL5901 thrashing speeds……………………………...43

III. Observing effects of longer exercise and food restriction treatments upon native
human ⍺-synuclein species………………………………………………………………47

IV. Summary: What both shorter and longer bouts of exercise and food restriction can
tell us about the differential effects of these treatments upon native human ⍺-synuclein
protein species………………………………………………………….………………...68
ix

Chapter 3: Observing the downstream effects of exercise or food restriction upon
NL5901 worms………………….……………………………………………………………….53

I. Analyzing thrashing behavior of previously exercised or food restricted worms……..53

II. Analysis of ⍺-synuclein protein species 1 hour after exercise or food restriction
treatments……………………………………………………………………….………..60

III. Analysis of ⍺-synuclein protein species 24 hours post exercise or food restriction
treatments………………………………………………………………………………...64

IV. Summary: Observing downstream effects of exercise and food restriction…………68

Chapter 4: Investigating the effects of pharmacological interventions on C. elegans
NL5901 and GMC101 worms…………………………………………………………….……..69

I. Investigating the role of protein degradation pathways under exercise and food
restriction conditions………..…………………………………………………………....69

II. Exploring the therapeutic effect of a novel HIF-1⍺ stabilizing compound,
Diphyllin (DP)……………………………………………………………………...……77

III. DP treatment of C. elegans models of Parkinson’s and Alzheimer’s
proteotoxicity………………………………………………………………………...…..89

IV. Summary: Exploring mechanisms involved in exercise and food restriction and
utilizing a novel natural product compound in proteotoxicity models……….…..……...96

Chapter 5: Future directions……………………………………………………………………...98
References ...................................................................................................................................102
Appendices...................................................................................................................................112
Appendix A: Methods..................................................................................................................112

*Data from chapters 1-3, specifically BN-Page results, confocal data, and thrashing assays, were
included in a publication published under the title “Swimming exercise reduces native ⍺-
synuclein protein species in the transgenic NL5901 C. elegans model of Parkinson’s disease,”
(Schmidt et al.)
x
List of Figures:
Schematic 1. Summary of key points of overlap between exercise and Parkinson’s…...………...9

Schematic 2: Representation of current hypothesis…………………………………………..….76

Schematic 3. Summary of differential effects between exercise and food restriction……...…....98

Figure 1. 15-20 minutes of swimming exercise (Ex) decreases native human
⍺-synuclein protein species………………………………………………………………………24

Figure 2. Representative western blot images showing the predominant
species present after exercise.……………………………………………………………………28

Figure 3. Representative confocal images of ⍺-synuclein puncta in NL5901 worms.…………..30

Figure 4. Exercise and food restriction significantly reduce total puncta #
per worm and average puncta surface areas (SA)………………………………………………..31

Figure 5. Individual puncta show a statistically significant difference between
exercised and food restriction conditions.……………………………………………………….34

Figure 6. Comparing analysis of three puncta size groups..…….……………………………….36

Figure 7. Exercise shows a progressive reduction in protein quantity every 5 minutes, and the
emergence of a single predominant band at 15 minutes of exercise.…………………………….41

Figure 8. Preliminary results from a timed experiment show the quantification of thrashing
rates in NL5901 worms at 15-minute intervals as well as corresponding samples from
SDS gel…………………………………………………………………………………………..45

Figure 9. Effects of 1 and 2 hours of exercise and food restriction.……………………………..49

Figure 10. Thrashing assay results on Day 2 (24 hours post-treatment) at the 3 minutes.……....56

Figure 11. Thrashing assay results after 20 minutes of exercise on Day 2 (24 hours post-
treatment)………………………………………………………………………………………...58

Figure 12. Thrashing assay results on Day 4 (96 hours post-treatment)………………………...59

Figure 13. Re-feeding (RF) worms for 1 hour post 15-20 minutes of exercise or food
restriction causes a significant increase in ⍺-synuclein protein species.………………………...62

Figure 14. Re-feeding (RF) worms for 24 hours after 15-20 minutes of exercise or food
restriction……………………………………………………………………………………...…65
xi

Figure 15. Pre-treatment of NL5901 worms with MG132 abrogates effects of food
restriction, but not exercise, on 66-<480 kDa range proteins.……………………...……………71

Figure 16. NAC pre-treatment shows a significant abrogation of the effects of food
restriction on 66-<480 kDa kDa ⍺-synuclein protein species as well as changes in the
protein profile of exercised worms.……………………………………………………………...74

Figure 17. Identification of Diphyllin (DP) as a HIF-1⍺ protein stabilizing natural product
and downstream protective effects.………………………………………………………………80

Figure 18. Preliminary results showing that 3 hours treatment with Diphyllin (DP) in
NL5901 worms significantly reduces the quantity of well proteins..………..…………………..91

Figure 19. Diphyllin (DP) significantly improves the thrashing ability of NL5901 worms.……94

Figure 20. Diphyllin (DP) protects GMC101 worms against paralysis at 48 hours post
treatment.………………………………………………………………………………………...95

xii
List of Abbreviations:

6HD: 6-Hydroxydopamine
A-β: Amyloid beta

⍺-synuclein: Alpha-synuclein
⍺-synuclein::YFP: ⍺-synuclein tagged to YFP
APA: Anticipatory Postural Adjustments

ARTI: Adapted Resistance Training with Instability

ATP: Adenosine Tri-Phosphate
BDNF: Brain Derived Neurotrophic Factor

BBB: Blood-Brain-Barrier
BNIP3: BCL2 Interacting Protein 3

BN-Page: Blue-Native Page
BOLD: Blood Oxygen Level Dependent Activation

CLR: Cerebral Locomotor Region

CP: Ciclopirox

DMSO: Dimethyl Sulfoxide

DP: Diphyllin

DSS: Disuccinimidyl Suberate

ENS: Enteric Nervous System
EPO: Erythropoietin

Ex: Exercise
ER-UPR: Endoplasmic Reticulum Unfolded Protein Response

xiii
Fe
2+
: Ferrous Iron

ftn-1: Ferritin

FOG: Freezing of Gate
FR: Food Restriction
FUdR: 5- Fluoro-2′-deoxyuridine

GFP: Green Fluorescent Protein
GI: Gastrointestinal Tract
GMC101: C. elegans worm strain containing A-β in muscle cells

HO-1: Heme-Oxygenase 1

HIF-1⍺: Hypoxia Inducible Factor-1 alpha
HIF-1β: Hypoxia Inducible Factor -1 Beta

HSP70: Heat Shock Protein 70

HRE: Hypoxia Response Element

HTS: High-Throughput Screen
KRS: Kufer-Rakeb Syndrome

ldh-1: Lactate Dehydrogenase

L-DOPA: L-3,4Ddihydroxyphenylalanine
MG132: Carbobenzoxy-Leu-Leu-leucinal

mM: Milli Molar Concentration

MITG: Middle and Inferior Temporal Gyrus

mito-UPR: Mitochondrial Unfolded Protein Response

MLR: Mesencephalic Locomotor Region

MPP+: 1-Methyl-4-Phenylpyridinium ion
xiv
MPTP: 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAC: N-Acetyl-l-Cysteine

NL5901: C. elegans worms strain containing ⍺-synuclein::YFP in muscle cells

NO: Nitric Oxide
NPL 640: Natural Product Library
OXPHOS: Oxidative Phosphorylation
PD: Parkinson’s Disease

PFA: Paraformaldehyde

PGC-1⍺: Peroxisome Proliferator-Activated Receptor-Gamma coactivator -1Alpha

PHD2: Prolyl Hydroxylase Domaine Protein 2
PNS: Parasympathetic Nervous System
PQ: Paraquat
PwP: Person with Parkinson’s
RF: Re-Feeding
RNS: Reactive Nitrogen Species
ROS: Reactive Oxygen Species
SA: Surface Area

SDS: Sodium Dodecyl Sulfate

SN: Substantia Nigra
SCFA’s: Short-Chain Fatty Acids
SIRT1: Sirtuin 1

SMA: Supplementary Motor Area

xv
SNpc: Substantia Nigra Pars Compacta
SNS: Sympathetic Nervous System
TH: Tyrosine Hydroxylase
TMR: Traditional Motor Rehabilitation

uM: Micro Molar Concentration

UPDRS: Unified Parkinson’s Disease Rating Score
VEGF: Vascular Endothelial Growth Factor

VHL: von Hippel Lindau

WPC: World Parkinson’s Congress
YFP: Yellow Fluorescent Protein

xvi
Abstract:
Exercise has been historically recommended to prevent many disease conditions. Intense
exercise in particular has been shown to be beneficial for Parkinson’s disease (PD) — stopping
and even reversing symptoms in some patients. Recent research in mammalian animal models of
Parkinson’s have shown that exercise affects ⍺-synuclein aggregate species, considered to be a
hallmark of PD. However, the exact changes in native ⍺-synuclein protein species after exercise
and the downstream effects of exercise upon the health of the animals remains unclear. Recently,
it was shown that swimming constitutes a form of exercise in C. elegans worms that confers a
protective effect in several worm models of tau and Huntington protein neurodegeneration. Here
we show that a period of swimming exercise (Ex) — 15-20 minutes — dramatically reduces
several native human ⍺-synuclein protein species in the NL5901 C. elegans worm model of
Parkinson’s. Exercise on Day 1 of adulthood was found to improve motor function measured by
the thrashing rate of worms on Day 2 and Day 4 when compared to both control (untreated) and
food restricted (FR) worms. Moreover, exercised worms show smaller ⍺-synuclein::YFP puncta
on average than food restricted worms as measured by confocal microscopy, as well as
significantly less higher molecular weight proteins as measured by BN-Page assays. Here we
show that exercise reduces native human ⍺-synuclein levels independent of food restriction in C.
elegans NL5901 worms.

xvii
Preamble.
Parkinson’s disease (PD) is a complex biological condition for which, currently, we do
not know the root cause or cure. In our search for how to best approach this problem, we drew
much inspiration from the experiences of those who have Parkinson’s. In particular, our
approach has been greatly guided by the empirical observations revealed to us by many People
with Parkinson’s (PwP), in a non-clinical/medicinal environment, by those who are close to us,
and those who discuss their observations and thoughts in public forums. Among our many
experiences, we were very fortunate to hear directly from PwP at the 5
th
World Parkinson’s
Congress (WPC) in Kyoto, Japan in June of 2019. Every person we know who has Parkinson’s
expresses such great focus and determination in their battle with this condition, while
simultaneously living in harmony with their situation. They truly adhere to the great adage
“without light, there is no darkness, and without darkness, there is no light.”
Through listening to and observing Parkinson’s symptoms, we have attempted to convert
our intuitive understanding of PD — the outward observations — into a concrete hypothesis of
what may be happening at the biochemical level. In particular, we were very interested in two
natural disease altering interventions. One approach is the participation in physical exercise — it
is not uncommon to hear a PwP say that they “feel like [they] no longer have Parkinson’s” after
exercising. The other is the improvement of gastrointestinal (GI) symptoms through dietary
changes, or other interventions. In our overarching approach to PD, we have attempted to marry
these two approaches — we wondered if exercise can affect the gastrointestinal system and, in
this way, perhaps alleviate the disease condition and halt progression. In our pursuit of this
intuitive approach, we are grounded in the vast, creative, and invaluable knowledge contributed
xviii
to this problem by the plethora of scientists working to solve Parkinson’s, as well as similar
questions.
In initially trying to understand how we can tackle Parkinson’s, we were inspired by Dr.
Gary Scharpe’s empirical observations. Dr. Scharpe, who has Parkinson’s himself, writes and
shares his thoughts about his symptoms on his website: OutThinkingParkinsons.com. One of his
posts in particular, a discussion of the role of the vagus nerve/parasympathetic nervous system
(PNS) and its relationship to Parkinson’s disease, caused our imagination to soar.
The vagus nerve, commonly referred to as the “gut-brain axis,” is a term that has
generated great attention in the last few years, and we believe, rightly so! Interest in the vagus
nerve has helped to bring forth a better understanding of the importance of GI health in
relationship to brain health, where disruption of the former has been linked to a variety of
conditions, including depression, Parkinson’s, and Alzheimer’s. Indeed, it is possible that we
have for far too long ill-considered the health of the gut in our overall well-being. In fact, it was
only approximately two-decades ago that the gastrointestinal system was found to contain its
own, relatively autonomous, nervous system — the enteric nervous system (ENS.) It may be
possible that the GI is truly a major source of both health and disease, and we are excited about
the many directions which research on the gastrointestinal system and its relationship to the brain
may take in the future.
In regards to Parkinson’s specifically, problems in the GI can be considered an early, but
not diagnostic, sign of disease onset. Decades before visible motor symptoms manifest, the
majority of PwP report experiencing some form of gastrointestinal disturbance, specifically
constipation. The high probability of this occurrence supports the real need for a biomarker,
which may help to predict, and possibly alter, the course of disease progression through
xix
administration of early interventions. In particular, Dr. Heiko Braak, a Parkinson’s specialist,
corelated observations of early GI symptoms and the progression of motor symptoms, to ⍺-
synuclein aggregate spread. He hypothesized that ⍺-synuclein aggregation may originate in the
periphery, such as the GI or nasal canal, and travel through the vagus nerve to the central
nervous system, including the Substantia Nigra (SN.) The SN, located in the midbrain, is the
locus of dopamine production and therefore, responsible for voluntary movement. In
Parkinson’s, it is the SN which suffers the greatest neuronal degeneration, and it has been
hypothesized that ⍺-synuclein aggregates are some of the major contributors, causing a
disturbance of cellular homeostasis and neuronal cell death. As such, it is possible that one
approach to alleviating Parkinson’s may be through therapeutic interventions aimed at reducing
⍺-synuclein aggregation and it is here that we wondered if exercise may play a role.
As mentioned before, exercise has shown great promise as a natural and strongly
effective therapy for PD. Interestingly, the vagus nerve plays an important role in this process as
a regulator of the sympathetic nervous system (SNS). While the SNS is responsible for allowing
the body to move while exercising, the vagus nerve/PNS is responsible for allowing the body to
achieve equilibrium afterwards. Furthermore, exercise has been shown to improve the strength of
the vagus nerve, while ⍺-synuclein aggregates diminish its function (Gladwell et al. 2010; Kai et
al. 2016; Musgrove et al. 2019; Sijben et al. 2020). During SNS activation, the gastrointestinal
system experiences a dramatic physiological change — blood is re-allocated away from the GI
tract towards tissues that are required for movement (heart, lungs, and muscles). It is therefore
possible that the changes taking place in the GI tract and vagus nerve in response to exercise may
help improve Parkinson’s symptoms, possibly through effecting changes to ⍺-synuclein
xx
aggregation. With these observations in mind, we initiated experiments to further understand if
the restorative effects of exercise may be due to alterations in ⍺-synuclein aggregation.
In order to simplify this complex question, we chose to use as a model the NL5901 C.
elegans worm model of Parkinson’s. Here, human ⍺-synuclein protein species are overexpressed
within the muscle cells, leading to aggregate formation and therefore a readily visible
phenotypical change in worm behavior i.e. slower thrashing rates. Our goal was to initiate novel
experiments exploring exercise in the context of PD. We are greatly thankful to Drs. Ricardo
Laranjeiro and Monica Driscoll from Rutgers University, whose incredibly comprehensive
research on exercise mechanisms in N2 (wild type) C. elegans worms propelled our work
forward. In the following chapters, we describe our observations of the effects of swimming
exercise upon the NL5901 worm model of Parkinson’s. We are eager to apply our findings
further in the context of a tangible therapeutic approach for Parkinson’s disease.

1
Introduction — Parkinson’s disease (PD) and exercise as a medicine.
Parkinson’s disease (PD) is the second most common neurodegenerative disease in the
world. It affects mainly the aging population between 60 and 80 years old, but in rarer cases, it
affects younger populations as well. Early onset PD begins in the late 40’s to mid 50’s, while
young onset PD begins in the 20’s to 30’s and is usually linked to genetic mutations. Although
several mutations have been described — SNCA, LRRK2, Parkin, ATP13A2 — Parkinson’s is
still not generally considered a genetic disease, especially in cases of normal (age-related) onset
(Klein and Westenberger 2012). Instead, PD appears to occur sporadically and evidence has
shown that exposure to environmental factors, such as heavy metals like iron, chemicals like 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or pesticides like paraquat and rotenone,
can cause Parkinson’s (Langston 2017; Shin et al. 2017). Indeed, these chemicals are used to
induce cell toxicity and neuropathology in cell and animal models of the disorder, mainly via
disturbances in mitochondrial function and increased oxidative stress (Tanner et al. 2011; Bus
and Aust 1976). Due to these connections several lawsuits, as well as a reform bill, have been
filed to prevent the agricultural use of paraquat based on data showing the prevalence of PD
amongst those exposed to it (Costello et al. 2009; Wang et al. 2011). Conversely, it is also
possible that genetic variations make some more individuals more susceptible than others to
these environmental factors.
Of the several non-motor symptoms in Parkinson’s, one of the most prevalent is the
experience of gastrointestinal disturbances (Salat-Foix and Suchowersky 2012). Here, it is
possible that exposure to chemicals via the gastrointestinal tract or nasal passageway may induce
⍺-synuclein aggregates, planting the seed for Parkinson’s early in the periphery (Chandra et al.
2017). In addition, shifts in gut microbial composition can occur, either on their own, or through
2
chemical or antibiotic interference. It was recently shown that gut microbiota play a role in
inducing ⍺-synuclein aggregate-mediated motor deficits in a mouse model of ⍺-synuclein
aggregation, possibly through the release of short-chain fatty acids (SCFAs) (Sampson et al.
2016). Furthermore, microbiota from Parkinson’s patients induced these same ⍺-synuclein
aggregate-mediated deficits in these mice. Interestingly, further studies showed that exposure of
mice to curli producing Escherichia Coli bacteria promoted ⍺-synuclein aggregation and
intestinal and motor impairments, further supporting the Braak Hypothesis (Braak et al. 2003;
Del Tredici and Braak 2016; Sampson et al. 2020). Interestingly, whereas aggregates were
previously only seen to affect the GI tract and vagus nerve/PNS before travelling to the brain,
they were also recently shown to spread to the SNS as well (Van Den Berge et al. 2019).
Outwardly, Parkinson’s is recognized by difficulty in regulating voluntary movement.
Patients can experience dyskinesia — large, jerky movements of the limbs, or tremors in the
hands or legs — and/or bradykinesia — a feeling of rigidity, where movements, particularly of
the arms and legs, can be difficult. PwP may also experience a symptom called freezing of gait
(FOG) — often cited as a feeling of “being glued to the floor” — as well as decreased control
over facial movement, often described as “the mask of Parkinson’s.”
These examples of reduced control over voluntary movement occur via a decrease in the
neurotransmitter dopamine, which is produced by dopaminergic neurons located in the SN.
Although it is thought that Parkinson’s begins decades earlier, today, a diagnosis can only be
made once a patient begins to experience some form of these physical symptoms. For example, a
small, persistent tremor in the hand, or some form of rigidity brings patients to see a neurologist.
However, a diagnosis can only be substantiated if patients respond to L-DOPA administration,
also known as “dopamine replacement therapy.”
3
Currently Available Medications:
L-DOPA (L-3,4-dihydroxyphenylalanine) is a natural precursor to dopamine, formed
through tyrosine hydroxylase (TH) conversion of L-tyrosine. It is converted to dopamine by L-
aromatic amino acid decarboxylase (Taylor and Creese). In its medicative form, L-DOPA is the
most effective Parkinson’s medication currently available, and is often referred to as “the gold
standard of Parkinson’s medication.” It works by replenishing insufficient levels of dopamine in
the body and is usually taken at an established dose at regular intervals throughout the day, such
as every four hours. This allows for a relatively constant level of dopamine to be present in the
body, with limited periods of interruption in voluntary movement. As with all medication,
however, there are side effects. In particular, it is difficult to establish the correct dosage for each
patient — while too little does not alleviate symptoms and can leave a PwP still feeling
immobile, too much can lead to dyskinesia, again causing a PwP to feel that they are not in
control of their body (Ashworth and Saunders 1985).
The need for L-DOPA specifically, as opposed to direct dopamine administration, lies in
its ability to cross the blood brain barrier (BBB). L-DOPA is also formulated with carbidopa, an
L-amino acid decarboxylase inhibitor, which helps prevent conversion of L-DOPA to dopamine
before it reaches the brain i.e. in the body’s periphery. Other medications, such as entacapone, a
catechol-O-methyltransferase-COMT inhibitor, also help to prevent the degradation of L-DOPA.
Entacapone is prescribed when a PwP begins to experience a worsening in “wearing-off”
symptoms, which occur when L-DOPA concentrations become low. This usually occurs near the
end of the four-hour medication cycle i.e. close to the time when the next dose needs to be taken.
Wearing-off symptoms can be described by the typical motor impairment present in PD,
but they may cause a PwP to experience a sometimes sudden change, from a relatively normal to
4
a perhaps sharply reduced functionality. These symptoms are decreased upon intake of the next
dose of medication, but relief is only sustained until the end of the next dose. Different
formulations of L-DOPA, such as rytary, are recent additions which provide more even levels of
medication throughout the day and do not require entacapone. While rytary contains the same
ingredients, it’s effectiveness comes from a different capsule formulation which provides a burst
of L-DOPA followed by a slow release (Mittur et al. 2017). This greatly helps to reduce the “on
and off” periods of Parkinson’s and increases functionality during the day. Interestingly, recent
research has shown that a burst of dopamine, instead of constant levels as provided by current
medications, may be all that is needed to initiate and sustain movement (da Silva et al. 2018).
This later data may help to inform future medical research aimed at easing Parkinson’s motor
symptoms.

Exercise as a medicine for aging and Parkinson’s:

Aging is considered to be the greatest risk factor in the development of Parkinson’s. A
recent study exploring lifespan extension effects in a C. elegans models of PD noted an
alleviation of Parkinson’s-related mechanisms including ⍺-synuclein aggregation in these
animals (Cooper et al. 2015). In general, research on the effect of exercise on aging have shown
strong benefits. For example, it was shown that five months of endurance exercise in a mouse
model of accelerated aging (progeria) not only reversed many visible phenotypes of aging such
as hair loss, but also improved other parameters such as endurance and mitochondrial processes,
even surpassing control levels (Safdar et al. 2011). A separate paper showed that a 6-month
resistance exercise regimen caused a reversal in the gene profile of older participants (60’s and
70’s), turning it towards the profile of young, relatively sedentary participants (20’s) (Melov et
al. 2007). In particular, the authors noticed that genes affected most by aging were those related
5
to mitochondrial function. Furthermore, the data interestingly showed that exercise preferentially
affects genes related to aging. This suggests that upregulating exercise mechanisms may help
alleviate aging and related conditions.
While there is no current formulated medication which can grant lasting relief from PD, it
has been shown that exercise can help alleviate many symptoms. If we pause and briefly
consider the complex physiological functions that are required for normal movement throughout
our day, we may feel even more overwhelmed when considering what occurs when we start to
move at faster pace and engage in more complicated maneuvers. Furthermore, if one considers
that PwP already struggle with the first without the addition of the second, one may wonder how
is it that exercise exhibits such an overwhelmingly high improvement of symptoms in those with
PD.

Similarities between exercise and Parkinson’s:
There appear to be several parallel mechanisms involved in both exercise and Parkinson’s
and perhaps better understanding their role will provide a deeper understanding of PD. Exercise
is mechanistically and physiologically complicated but it can be summarized by the following
stepwise points: After the cue for movement occurs, norepinephrine is released and acts to
reallocate blood flow towards organs innervated by the SNS — those that are required for
movement such as the heart, lungs, and muscles — and away from organs innervated by the
PNS, which are not required for movement — such as the gastrointestinal (GI) system, liver, and
pancreas. In this way, during exercise, the SNS is activated while the PNS is suppressed. After
exercise is ceased, the PNS re-activates and suppresses the SNS, thereby re-establishing
equilibrium.
6
In order to provide energy to cells while the body is in motion, glucose is mobilized and
converted into energy. If the body has been moving for more than 1 minute, cells metabolize
glucose through aerobic glycolysis followed by oxidative phosphorylation (OXPHOS) in the
mitochondria, to produce the energy molecule, adenosine tri-phosphate (ATP). During this
process, mitochondria produce the reactive oxygen species (ROS) — superoxide (O2
.-
), hydrogen
peroxide (H2O2), and the hydroxyl radical (HO
.
). ROS are high energy molecules, and as such,
interact with a variety of proteins and molecules in order to lower their own energy state. While
at high concentrations (oxidative stress) these reactions can lead to damage through changes in
protein and molecular structure and function, ROS are also important signaling molecules that
are involved in reversible post-translational modifications, such as the oxidation of methionine
residues, and are important actors in inducing autophagy upon nutrient imbalance, such as
depletion of glucose levels (Filomeni et al. 2015).
If we consider in parallel someone experiencing Parkinson’s, we come across several
points of similarity to mechanisms upregulated during exercise. While a PwP may not be able to
easily initiate movement, their symptoms, such as lowered vagus nerve (PNS) and GI
functionality, may indicate a heightened upregulation of the SNS. This may occur through
interference of nervous system function from the presence of ⍺-synuclein aggregates as
previously discussed (Van Den Berge et al. 2019). Furthermore, mitochondrial dysfunction has
been reported and closely tied to PD (Chen et al. 2020). Many PwP have also been cited to crave
sugar (Kennedy et al. 1994; Miwa and Kondo 2008). Although perhaps related to the dopamine-
reward system, this symptom may also point to an underlying energy deficit, specifically
cognitive lethargy, which is another non-motor symptom of PD (Stocchi et al. 2014; Friedman et
7
al. 2016). Given this, exercise may provide benefit via improvement of mitochondrial function as
previously cited (Melov et al. 2007; Safdar et al. 2011).
Another similarity between exercise and Parkinson’s lies in ROS production. It has been
shown that high ROS concentrations can lead to increased dopamine auto-oxidation, forming
dopaquinones and radical species (Dias et al. 2013). Furthermore, dopaquinones can cyclize to
aminochrome, a precursor to neuromelanin, which has been implicated in neuroinflammation,
and which can also form adducts that interact with proteins, including ⍺-synuclein. With
oxidative stress seemingly quite high in Parkinson’s already, it may seem counterintuitive that
exercise should confer a strong therapeutic effect because of its increase in ROS levels for some
time. This begs the question: would it make more sense to treat PD with anti-oxidants rather than
a source of oxidants, such as exercise?
This dichotomous relationship is not unique to Parkinson’s, however — marrying the
general benefits of exercise and caloric restriction to the concomitant generation of ROS
suggests that this stress is a form of hormesis (Kahn and Olsen 2010; Powers et al. 2020).
Furthermore, ROS and reactive nitrogen species (RNS) production can be protective in the
context of the ⍺-synuclein species present. It has been shown that oxidation of ⍺-synuclein at
methionine residues results in a protective species that inhibits aggregate growth and spread
(Uversky et al. 2002). In addition, nitrosylation of tyrosine residues similarly prevents the
elongation of fibrils and the spread of aggregation (Yamin et al. 2003). Interestingly, it was also
observed that exposure of ⍺-synuclein monomers or dimers to UV-induced ROS resulted in di-
tyrosine bonds within the dimeric species, which also served to protect against aggregate growth
and spread (Wördehoff et al. 2017). In contrast, exposure of ⍺-synuclein fibrils to the same ROS
source yielded di-tyrosine bonds that resulted in insoluble and pro-aggregative ⍺-synuclein
8
species. This highlights that protection against ⍺-synuclein aggregation can be achieved through
treatment with ROS, but that the correct initiating ⍺-synuclein species are an important source of
this effect.
The detrimental chemical modifications due to ROS have also been put into question in
the “radical free” concept, which brings to light the reversible nature of ROS reactions with
cysteine and methionine residues that are regulated by methionine sulfoxide reductases (MSR),
and highlights the potential role of ROS and RNS as signaling molecules which can function as
switches to change protein functionality, for example (Filomeni et al. 2015). Interestingly, recent
research has also observed that feeding the NL5901 C. elegans worm model of Parkinson’s the
probiotic Bacillus Subtilis, resulted in protection against ⍺-synuclein aggregation partially
through the ability of this bacteria to produce the RNS species, nitric oxide (NO) (van Ham et al.
2008; Goya et al. 2020). Taken together, these data may suggest that generation of ROS and
RNS during exercise can confer protective effects through formation of ROS and RNS modified
⍺-synuclein protein species.
9

Schematic 1. Summary of key points showing overlap between exercise and Parkinson’s
mechanisms.

Clinical and cohort studies demonstrating effects of exercise upon PwP:

Overall, there is a strong consensus about the benefits of movement in PD (Uhrbrand et
al. 2015). Walking exercise as a positive habitual activity was even mentioned in Dr. James
Parkinson’s An Essay on the Shaking Palsy (1817), the first comprehensive description of
Parkinson’s disease (Parkinson 2002). However, one of the earliest papers mentioning specific
recommendations for exercise was in a 1954 paper beautifully titled “Physical Medicine and
Rehabilitation for the Elderly Neurologic Patient,” which discusses certain stretches and
strengthening exercises of the extensor muscle groups (required for straightening limbs)
(Watkins 1954). Interestingly, the author cautions that these exercises should be confined within
the extremes of fatigue.
10
While fatigue is not expressly defined in this article, it likely refers to exhaustive, or more
intense, forms of exercise. In contrast to this suggestion, a 2017 Phase II clinical trial showed
that high intensity treadmill exercise — 80-85% heart rate capacity, 30 minutes a day, 3-4 times
a week, over the course of 6 months — was more beneficial to PwP than moderate or low
intensity exercise (Schenkman et al. 2018). In this study, patients experienced improvements in
several parameters including an 8% increase in oxygen consumption (VO2 max), an indicator of
aerobic capacity. Importantly, those enrolled in the high intensity program exhibited a significant
reduction in disease progression. For example, while the moderate and untreated groups
experienced a 2.0 and 3.2-point increase in the Unified Parkinson’s Disease Rating Score
(UPDRS), a calculation of physical symptoms attributed to Parkinson’s and a measure of disease
progression respectively, the high intensity group experienced only a 0.3-point increase. It is also
important to note that this trial was performed in newly diagnosed Parkinson’s patients who were
not yet taking anti-Parkinsonian medications including carbidopa/L-DOPA. This data suggests
that if those who are recently diagnosed become involved in high intensity activity early on, it is
possible to significantly alter the course of the disease. In addition, while it is important to
always be cautious, this study showed that high intensity treadmill exercise is tolerable and safe
for PwP, highlighting accessibility as a treatment. These results have prompted further
investigations in a Phase III clinical trial which was initiated on January 1, 2021.
A recent study also focused on the effects of a physical therapy regimen aimed at
addressing freezing of gait (FOG) symptoms (Silva‐Batista et al. 2020). FOG occurs when PwP
display repeated anticipatory movements, such as repeated movements to lift one leg to take a
step, but then are not able to release the leg to complete the movement. In other words, one
wants to initiate a movement, but one does not know how to complete it. Those with FOG have
11
been shown to have reduced blood oxygen level dependent (BOLD) activation in the
mesencephalic locomotor region (MLR), located in the midbrain, associated with the initiation
and control of movement. In order to address this, the authors initiated a 12-week physical
therapy regimen aimed at simultaneously activating different parts of the brain. Specifically, the
authors focused on adapted resistance training with instability (ARTI) which requires “high
cognitive, proprioceptive (perception or awareness of the position of the body) and motor control
demands,” which were adapted to include exercises that involve anticipatory postural
adjustments (APA), such as lunge exercises. Participants were compared to a control exercise
group participating in traditional motor rehabilitation (TMR), which involves lower complexity
exercises. The study outcomes showed that higher complexity exercises improved BOLD in the
three out of four areas shown to be affected by FOG: the middle and inferior temporal gyrus
(MITG), mesencephalic locomotor region (MLR), and the cerebral locomotor region (CLR.) The
authors did not see a significant difference in the supplementary motor area (SMA.) Overall, the
study found that ARTI was likely effective because it involved exercises that 1) incorporated
movements emulating issues encountered during FOG, 2) increased complexity by performing
exercises on unstable surfaces thus improving visual sensory perception, 3) improved postural
perception, and 4) increased automaticity.
Several studies have also observed the beneficial effects of other forms of lower-impact
exercises such as yoga, Tai Chi, and dance (Feng et al. 2020). A recently published work
observed the effect of weekly dance classes that also engaged a variety of different aspects of
concern for PwP including endurance, cognition, rhythm, balance, and depression, and is the first
study to show a lack of decline in motor and non-motor PD symptoms in a three-year
preliminary longitudinal study (out of ten years) (Bearss and DeSouza 2021). This supports the
12
idea stressed above — that engagement of different parts of the brain during an exercise regimen
can yield great benefit to PwP.

⍺-synuclein in Parkinson’s neuropathology:
While there are many mechanisms upregulated during exercise which improve
mechanisms related to Parkinson’s, we became interested in studying ⍺-synuclein aggregation
specifically. While ⍺-synuclein is present in brain synapses and is also expressed in almost all
cell types, its physiological role is still not certain. ⍺-synuclein is a 14 kDa protein belonging to
the family of intrinsically disordered proteins. As such, it is able to interact with a large array of
chemicals (Mor et al. 2016). Furthermore, ⍺-synuclein aggregation is also not very well
understood and, while it has been shown to be detrimental to cellular health, it also has been
suggested to play a role in quarantining pathogens or harmful chemicals (Lashuel et al. 2013;
Chen et al. 2016; Barbut et al. 2019). Recently, it was also shown that ⍺-synuclein remains in the
monomeric state 1,000 times longer than previously reported before assuming different forms of
interactions and conformations including: the protective tetrameric state, the proposed toxic
dimeric state, different fibrillar states, and various interactions with lipids (Chen et al. 2021
May).
Recently, it has been suggested that lipid molecules may play an important part in
Parkinson’s neuropathology (Shahmoradian et al. 2019). Here, it was observed that the
components of Lewy Bodies, long thought to contain mostly large inclusions of ⍺-synuclein
fibrils and aggregates, appear to mainly contain lipid vesicles with a high concentration of non-
fibrillar ⍺-synuclein forms. Recent work has also shed light on interactions between ⍺-synuclein
and lipid vesicles and the discovery of various structural patterns — namely ribbons, waves,
13
helices, and tight helices — in the presence of lipids (Meade et al. 2020). The role of ⍺-synuclein
in synaptic vesicles has also been of great interest, and it was recently found that ⍺-synuclein
interacts with the inner presynaptic plasma membrane where it allows synaptic vesicles to dock
(Man et al. 2021). Here, it was also shown that disruption in the lipid structure affected this
process, highlighting the importance of maintaining lipid membrane structures, which are known
to be disrupted in Parkinson’s.
In addition, Perez et al. discussed an interesting role of monomeric ⍺-synuclein as a
regulator of tyrosine hydroxylase activity (Perez et al.). As mentioned previously, TH catalyzes
the conversion of L-tyrosine to L-DOPA. Here it was shown that monomeric ⍺-synuclein
contains a sequence homology to chaperone protein 14-3-3, which serves to regulate TH. In this
regard, ⍺-synuclein’s homology to 14-3-3 allows it to bind to and regulate TH activity and
therefore dopamine production. Consequentially, ⍺-synuclein aggregation may lead to de-
regulated L-DOPA production and sequentially, greater dopamine synthesis. While this process
may be harmful if concentrations of dopamine are too high (it is a very reactive chemical that
can be easily oxidized forming radical species), it may also point to a physiological role for ⍺-
synuclein oligomers. It was shown that ⍺-synuclein can indeed form physiological tetramers,
shown to be expressed in various cell types (Bartels et al. 2011). In addition, these species were
resistant to aggregation. Altogether, this research sets a precedent for further investigation of the
physiological role of ⍺-synuclein oligomers.

Animal models of exercise explore benefits for Parkinson’s:
To this date, research into exercise as a therapeutic for PD has mainly focused on its
effect in reducing ⍺-synuclein aggregate species including monomers, oligomers, and
14
aggregates. A recent paper showed that three-months of voluntary running wheel exercise in a
mouse model of ⍺-synuclein aggregation of diffuse Lewy Body disorder (a Parkinson’s plus
disorder), significantly reduced the presence of dimers and oligomers in the brains of exercising
mice (Zhou et al. 2017). The study also found an increase in brain derived neurotrophic factor
(BDNF), a neuroprotective protein, as well as heat shock protein 70 (HSP70), known for helping
guide the proper folding of proteins (Mayer and Bukau 2005; Bathina and Das 2015). Through
the exercise regimen, researchers also observed an increase in DJ-1. While the function of DJ-1
is still not clear, mutations have been shown to cause autosomal recessive Parkinson’s (Zhou et
al. 2017; Repici and Giorgini 2019). Interestingly, the authors noted that DJ-1 knockout mice
had significantly (about 5-fold) lower running distances than wild-type mice. The authors also
observed an increased presence of monomers and dimers within the blood plasma of exercised
mice and have hypothesized that ⍺-synuclein aggregates were cleared from the brain into the
blood stream, possibly by the excretion of ⍺-synuclein proteins through exosomes from neurons.
Interestingly, in a separate work, it was shown that bi-directional transfer of ⍺-synuclein species
from the gut to the brain, and from the brain to the gut, occurs through blood circulation
(Arotcarena et al. 2020; Sgambato 2020). Altogether, these data suggest that assessment of levels
in the blood may be an effective biomarker to track alleviation of ⍺-synuclein aggregation in the
brain through exercise.
Several other studies utilized chemically induced mouse and rat models of Parkinson’s
disease in conjunction with forced or otherwise more controlled methods of treadmill exercise. A
recent paper showed that in a rotenone-treated rat model of Parkinson’s, while rotenone
significantly induced ⍺-synuclein staining in the substantia nigra and striatum, treadmill exercise
restored levels back to control. In addition, the exercised animals showed improved results in the
15
rotarod behavioral assay (Shin et al. 2017). A different study utilizing the MPTP model of
Parkinson’s found that ⍺-synuclein monomer levels induced by MPTP treatment, were
attenuated in the Substantia Nigra pars compacta (SNpc) as well as the striatum, after eight
weeks of treadmill exercise (Koo and Cho 2017). In addition, apoptotic cell death due to MPTP
treatment was reduced and TH, SIRT1 (implicated in aging), and PCG-1⍺ (involved in energy
metabolism) protein levels were increased. Treadmill exercise has also been shown to improve
autophagic flux as determined by a significant decrease in p62 levels. Conversely, a different
paper showed that while a four-week treadmill exercise program improved gait and postural
stability in a mouse model of human ⍺-synuclein overexpression as well as reducing levels of ⍺-
synuclein oligomers, there was no increase in cerebral autophagy (Minakaki et al. 2019). This
highlights the fact that there are still questions that need to be answered about the mechanisms
involved in order to understand the effects of exercise on ⍺-synuclein protein species.
Altogether, this research shows the powerful abrogative effect of exercise on different
Parkinson’s neuropathological hallmarks using different exercise methods in various mouse and
rat models. This research also shows that exercise invokes various neuroprotective pathways and
taken together confirm clinical observations regarding the benefits of exercise in human trials in
PwP.
In utilizing animal models, it is important to consider that animals engage in a significant
amount of physical activity as part of their survival habits. Therefore, understanding the effects
of movement and what constitutes exercise in various model organisms can perhaps hold the key
to answering many questions. While the mouse or rat mammalian system is close to that of
humans in many respects, it’s complexity can serve as a source of difficulties in data
interpretation. The relatively simple C. elegans worm, which contains approximately 83%
16
homology to mammalian systems, provides an alternative to better understanding the
mechanisms underlying the beneficial effects of exercise on PD.
Recent research into exercise mechanisms in the N2 C. elegans worm show that
swimming invokes similar mechanisms upregulated during mammalian exercise (Laranjeiro et
al. 2017). Here, the authors found that worms swimming for a single bout lasting 1.5 hours
expended more energy than sedentary worms. In addition, swimming exercise invoked
mechanisms of oxidative stress (such as an increase in anti-oxidant mechanisms) as well as
increased heat shock responses (including HSP70), the mito UPR, the ER UPR, and the hypoxic
response via increased lactate dehydrogenase (ldh-1) gene transcription. In addition, between 1
and 4 hours after exercise, it was shown that a significant reduction occurred in various genes
involved in glucose metabolism, indicating glucose mobilization during swimming exercise.
Glucose is similarly mobilized in humans during increased SNS activity such as during exercise
(Bear et al.)
Having established that swimming constitutes exercise in the C. elegans worm, the
authors optimized a single bout exercise protocol. Here, worms were subjected to 1.5 hour
sessions of swimming exercise several times a day in the first four days of adulthood e.g. three
times a day in the first two days of adulthood and two times a day in the next two days of
adulthood, with four hour breaks in between during which worms rested and biochemical
mechanisms achieved equilibrium (Laranjeiro et al. 2019). Using this regimen, the authors
measured the effect on ten genes related to muscle function and compared the results to less and
more intense schedules i.e. exercise one or two times a day versus four times a day. The authors
found that while the less intense schedules induced small or insignificant changes in muscle
genes, the more intense exercise regimes decreased expression of several genes, and therefore
17
were likely detrimental for the worms. Just like the approach in selecting the best drug dose or
medical treatment, this data highlights the importance of utilizing the correct ‘dose’ of exercise
for achieving the best health benefits. Utilizing this optimized regimen, the authors tested and
observed improvements in worm neuromuscular strength, abrogation of gut permeability at Days
11 and 15 of adulthood, extension in median lifespan, and neuroprotection in models of
Huntington’s disease and tau aggregation.

Glucose restriction in the context of proteotoxicity:
Alongside exercise, caloric restriction — the reduction of certain food consumption —
has been shown to extend lifespan in many model organisms — from yeast to mammals — and
confer protection in various diseases from cardiovascular to neurodegenerative (Mattson et al.
2017). Interestingly, it has also been shown that exercise and caloric restriction can have varying
effects on protein aggregation. In a model of tau pathology in obese mice, human tau
overexpressing mice were fed a high fat diet (Western) and given a sugary water to drink
containing a 42-44% serving of glucose, among other sugars. The study then observed the effect
of two interventions — voluntary running wheel exercise and caloric restriction through a
healthier diet. Interestingly, while two months of running wheel exercise reduced tau
phosphorylation, animals treated with a calorie restricted diet showed an increase in insoluble tau
(Gratuze et al. 2017). While it was pointed out by the authors that the presence of insoluble
proteins may not necessarily be detrimental — perhaps serving to protect against pathogens or
toxic chemicals — the strong change which this diet induced in tau proteins and its deviation
from the effects of exercise, is certainly a very interesting and important point for consideration.
In addition, in attempting to better understand the underlying mechanisms, the authors explored
18
the roles of protein kinases, the insulin pathway (found to be involved in regulating tau), as well
as proteasomal degradation and autophagy. However, they were unable to implicate any of these
pathways on the noted changes in tau.
In support of this data, it has been shown that while reducing glucose consumption can
extend the lifespan of C. elegans worms, treating worm animal models of Huntington’s, ALS,
and Alzheimer’s with glucose appeared to alleviate proteotoxicity (Tauffenberger et al. 2012).
Furthermore, it has been shown that glucose deprivation in human dopaminergic SHSY5Y cells
leads to increased aggregation of ⍺-synuclein proteins (Bellucci et al. 2008). Here, the authors
suggested that reduced glucose levels alter mitochondrial function, leading to decreased ATP
levels and increased levels of ROS and oxidized products, such as the previously mentioned
dopaquinones, which have been show to induce ⍺-synuclein aggregation. In addition, it was
shown that glucose deprivation resulted in increased levels of ⍺-synuclein proteins in general,
which is known to promote aggregation. Altogether, this research suggests a very interesting
relationship between glucose and ⍺-synuclein and new questions surrounding recommendations
of caloric restriction in the context of proteotoxicity.

The protective effects of the Hypoxia Inducible Factor (HIF-1⍺) in Parkinson’s:
It has been shown that lysosomal function, the key organelle responsible for breaking
down cellular components during autophagy, decreases with age (Bourdenx and Dehay 2016;
Nixon 2020). Furthermore, autophagic mechanisms are reduced in Parkinson’s and cause the
accumulation of ⍺-synuclein aggregates. Previously published work from the Andersen
laboratory suggests that neuronal hypoxia inducible factor – 1 alpha (HIF-1α) induction may
constitute a promising novel target for the prevention or slowing of neurodegeneration associated
19
with Parkinson’s disease (PD) (Rajagopalan et al. 2016). In particular, it was found that the
lysosomal p-type ATPase, ATP13A2, which is known to be mutated in a young-onset form of
PD called Kufer-Rakeb Syndrome (KRS), contains a hypoxia response element (HRE). Recently,
ATP13A2 was implicated in polyamine uptake through endocytosis and release into the cytosol,
where de-regulation leads to lysosomal mediated cell death (van Veen et al. 2020: 2). In addition
to ATP13A2, HIF-1⍺ induces many other downstream targets, including target genes involved in
neuroprotection and neurogenesis — such as the vascular endothelial growth factor (VEGF),
erythropoietin (EPO), and heme-oxygenase 1 (HO-1.) In addition, stabilization of HIF-1⍺
protein levels induce BCL2 Interacting Protein 3 (BNIP3), an autophagy and mitophagy
regulating gene. Therefore, among its many roles, HIF-1⍺ may lead to alleviation of lysosomal
dysfunction and increased autophagic clearance.
On its own, HIF-1⍺ is a highly regulated protein that is stabilized under hypoxic (low
oxygen) concentrations. In the brain, HIF-1α is regulated by PHD2 (Prolyl Hydroxylase Domain
2), an oxygen sensor, which binds to molecular oxygen under normoxic (normal) oxygen
concentrations (approximately 5% oxygen in the body). In addition, PHD2 also interacts with
cofactors ferrous iron (Fe
2+
) and α-ketoglutarate. Under normoxic conditions, PHD2 interacts
with HIF-1α and, in the presence of its cofactors, hydroxylates the proline residue of HIF-1α first
at position 564, and at higher oxygen concentrations, at position 402 (Chowdhury et al. 2016).
Hydroxylated HIF-1α is then recognized by the von Hippel Lindau (VHL) protein, subsequently
ubiquitinated, and degraded via the proteasome.
HIF-1⍺ has also been shown to be induced in GI tissues in response to exercise (Wu et al.
2020). As discussed earlier, the induction of the SNS during exercise leads to the re-allocation of
blood away from the GI tract towards organs required for movement. This physiological
20
response leads to hypoxia in gut epithelial tissues and the subsequent upregulation of HIF-1⍺.
Here, it is possible that increased HIF-1⍺ levels can lead to increased autophagy and ⍺-synuclein
aggregate clearance. Furthermore, ROS production in GI tissues also occurs in response to
hypoxia (Keirns et al. 2020). As such, ROS may induce the formation of the previously
discussed protective ⍺-synuclein species. In addition, ROS can also induce HIF-1⍺ (Chen et al.
2018). Therefore, ROS may mediate autophagy of aggregate species either directly as previously
discussed, or through increased HIF-1⍺ levels. Altogether, these mechanisms may suggest an
explanation for exercised induced benefits in the context of Parkinson’s disease.

Concluding remarks:
As discussed in this section, exercise has been shown to confer strong protective effects
in human clinical trials, as well as animal models, of Parkinson’s disease. As such, the
effectiveness of this natural therapy is very promising and understanding the mechanisms
involved may lead to a clearer and more directed approach to its application. As it was found that
swimming bouts in C. elegans constitutes a form of exercise similar to that observed in
mammals, we proceeded to examine the effect of exercise on native human ⍺-synuclein protein
species in the NL5901- pkIs2386 worm model of Parkinson’s (van Ham et al. 2008). In order to
better understand the effects of exercise upon the protein in its natural state and avoid any
possible modifications which may occur during work up, we performed tissue analysis via Blue
Native (BN) page westerns and confocal microscopy. In addition, we controlled for the effect of
reduced food intake by exposing worms in parallel to a period of food restriction (FR)
conditions. In the following chapters, we show that a short period of swimming exercise (Ex) —
15-20 minutes — dramatically reduces several native human ⍺-synuclein protein species in the
21
NL5901 worms directly after an exercise bout. Moreover, we demonstrate that the effects of
exercise are different from the effects of food restriction alone both in the short term and
downstream. We hypothesize that this divergence occurs through ROS mediated chemical
modifications of the distinct ⍺-synuclein species remaining after Ex and FR treatments.

22
Chapter 1. Effects of a short bout of swimming exercise on native human ⍺-
synuclein protein species.*

*Data from chapters 1-3, specifically BN-Page results, confocal data, and thrashing assays, were
included in a publication published under the title “Swimming exercise reduces native ⍺-
synuclein protein species in the transgenic NL5901 C. elegans model of Parkinson’s disease,”
(Schmidt et al.)

Parkinson’s disease (PD) patients have been shown to benefit greatly from intense
physical activity (Schenkman et al. 2018). Recent studies have demonstrated that exercise causes
changes in the levels of ⍺-synuclein aggregate species, a hallmark of PD, in different mammalian
animal models (Koo and Cho 2017; Shin et al. 2017; Zhou et al. 2017; Minakaki et al. 2019).
However, questions still remain about how exercise affects native ⍺-synuclein protein species
directly after the cessation of exercise and the longer-term downstream effects which exercise
may have on organismal health.
It was recently discovered that periods of thrashing in liquid solution, otherwise called
swimming exercise, in C. elegans worms induces many mechanisms invoked during mammalian
exercise (Laranjeiro et al. 2017). This has provided an avenue for studying exercise conditions in
various C. elegans models of neurodegeneration (Laranjeiro et al. 2019). Here, we describe the
effect of 15-20 minutes of swimming exercise and food restriction upon native ⍺-synuclein
proteins species in the NL5901 worm and further investigate the predominant ⍺-synuclein
protein species present after these treatments. We also discuss our results from confocal
microscopy experiments, which confirm the effects of exercise upon native ⍺-synuclein protein
species.

23
I. Effects of exercise and food restriction upon native human ⍺-synuclein protein species.

It has been previously reported that NL5901 worms exhibit a lower thrashing ability than
N2 worms due to the presence of ⍺-synuclein species in muscle cells (Anand et al. 2020). In our
experiments, we subjected Day 1 NL5901 worms to a period of swimming exercise — 15-20
minutes — and observed a dramatic effect upon the quantity of native human ⍺-synuclein
proteins via BN-Page westerns using an anti-GFP antibody to detect ⍺-synuclein::YFP proteins
as previously described (Figure 1 A-D) (van Ham et al. 2008). As worms continue to swim while
they are being collected for Western Blot assays, and because the speed of this collection can
vary, we have indicated the 15-20 minutes time frame in order to account for this variation.
Three different protein groups were analyzed — 66-<480 kDa, 720 kDa, and proteins trapped
within the wells (well proteins), which may be large molecular weight protein species. In the
exercised worms, 66-<480 kDa and well proteins were found to be significantly decreased
compared to control (un-treated) and food restricted worms.
24

Figure 1. 15-20 minutes of swimming exercise (Ex) decreases native human ⍺-synuclein protein
species. A. 66-<480 kDa range proteins in worms exercised for 15-20 min. on Day 1 of
adulthood are significantly reduced compared to control (un-treated) and 15-20 min food
restricted (FR) worms (***p£0.0001,

**p£0.0088). These proteins are also significantly reduced
25
for exercised versus food restricted worms (*p£0.0116.) B. Representative Blue Native (BN)
Page western blot showing the protein profile of control (un-treated), Ex, and FR worms,
demonstrating a reduction in proteins in 66-<480 kDa, 720 kDa, and well proteins. C. 720 kDa
proteins in Ex worms are significantly reduced compared to control (un-treated) and almost
significantly compared to FR worms (***p£0.0008, p£0.0548.) 720 kDa proteins are not
significantly reduced in FR worms (p=0.4301). D. Well proteins in Ex worms are significantly
reduced compared to control (un-treated) and FR worms (***p£0.0002, ****p<0.0001). Well
proteins are also significantly reduced in FR worms (*p£0.0144). N= 8, n=25 per replicate for A-
D.

Interestingly, we also observed that the protein profile in the 66-<480 kDa range was
different for exercised versus food restricted worms — while in exercised worms there appeared
only a single band for ⍺-synuclein at ~90 kD, in food restricted worms we detected a range of
protein similar to, but less abundant than those proteins present in control (un-treated) worms
(Figure 1B). For FR worms, of the three protein groups analyzed, only the 66-<480 kDa range of
proteins and the well proteins show a statistically significant reduction when compared to control
(un-treated) worms, while 720 kDa proteins do not show a significant decrease (Figure 1A and
C, respectively). This data illustrates the dramatic reduction in native ⍺-synuclein proteins
species due to just 15-20 minutes of swimming exercise, specifically demonstrating the effects of
exercise on all native ⍺-synuclein species immediately after exercise. Furthermore, our results
draw parallels to previous research which has shown a decrease in dimeric and oligomeric
species in a mouse model of diffuse Lewy body disorder after three months of voluntary running
wheel exercise, suggesting a viable comparison between the effects of exercise on ⍺-synuclein
proteins species in these different model organisms (Zhou et al. 2017).

II. Analysis of predominant ⍺-synuclein species present after exercise and food restriction
treatments.

26
Although proteins in BN-Page gels run according to molecular weight with the addition
of G-250 — a component which adds a net negative charge to proteins without changing the
native conformation — their three-dimensional structure may still influence at which molecular
weight they will appear in the gel. Consequentially, proteins which run through a BN-Page gel
may appear at a different molecular weight than in samples prepared for SDS based gels. Such
differences were previously observed for ⍺-synuclein monomers — while these species appear at
~14 kDa in SDS gels, they are present at ~50 kDa in BN-Page gels, indicating a substantial
difference in molecular weight as visualized by these separate methods (Bartels et al. 2011).
Previous research has suggested that submonomeric ⍺-synuclein protein species, which were
identified to be slightly lower in molecular weight than monomeric species as determined in SDS
gels, appear slightly above 45 kDa in BN-Page gels (Goya et al. 2020). However, the identity of
monomeric ⍺-synuclein species tagged to YFP within BN-Page gels has not yet been
determined.
In order to investigate the molecular weight of the predominant species that appears in
exercised worms as seen in BN-Page gels, we prepared samples in the presence of a crosslinker
— used in order to account for possible non-covalent interactions which may otherwise be
perturbed during work up — and analyzed them via SDS page gels. Here, we chose to work with
the disuccinimidyl suberate (DSS) crosslinker, which reacts with lysine residues and forms
bonds that are 8Å in length. DSS was utilized previously to link ⍺-synuclein and observe the
presence of physiological tetrameric ⍺-synuclein oligomers in various cell types (Bartels et al.
2011). Here, our preliminary results show that while control (un-treated) and food restricted
worms show a multitude of bands, exercised worms contain predominantly one band below 50
kDa, and a small quantity of well proteins (Figure 2.) The molecular weight of ⍺-synuclein-YFP
27
is equivalent to 41.8 kDa, and has been previously observed to appear below 50 kDa in SDS gels
from tissue extracts of NL5901 worms (van Ham et al. 2008; Goya et al. 2020). Altogether, this
information suggests that the species predominately present in exercised worms are monomeric
(Figure 2A.) It is important to note that while we see that, with slight variations, samples with
and without DSS appear to be similar overall, DSS treated samples contain several bands that are
closer together and less distinct from one another than in non-DSS treated samples (Figure 2B.)
This suggests that DSS effectively maintained some of the non-covalent bonds present between
like species.
Altogether, this data suggests that if the ~90k kDa protein species seen in BN-Page gels
is monomeric, then the 720 kDa species are 8-mers, and proteins contained within wells are >11-
mers. Due to the complexity of crosslinking chemistry however, future experiments will aim to
work more closely with DSS, or other methods, in order to investigate the structural identity of
these protein species in greater detail. Furthermore, these results indicate that since the
predominant species appears to be monomeric ⍺-synuclein-YFP, exercise clears almost all
higher molecular weight proteins after just 15-20 minutes.
28

Figure 2. Representative western blot images showing the predominant species present after
exercise. (A) Samples without DSS and (B) samples with DSS under control (un-treated),
exercised (Ex), and food restricted (FR) conditions. In both DSS and non-DSS treated samples,
Ex worms have a predominantly single band corresponding to monomeric species (red arrow)
and a very strong reduction in well proteins (orange arrow.) DSS treated samples appear to have
less distinct bands and proteins present in between bands (green and blue arrows, respectively.)
Blots were blocked with anti-GFP antibody. (N=2, n=25 worms per replicate.)

III. Observing ⍺-synuclein protein species in whole tissue using confocal microscopy.

i. Quantification of puncta number (puncta #) and average surface areas (SA)

29
Using confocal microscopy, we assessed the effect of exercise and FR on the surface area
(SA) size of ⍺-synuclein puncta located in the head region of the NL5901 worms as previously
reported (Goya et al. 2020) (Figure 3 A-F). Quantification of overall results indicate that both
exercised and food restricted worms contain a significantly lower number of puncta than control
(un-treated) worms (Figure 4A.) In addition, the average surface area of puncta is significantly
lower in both exercised and food restricted worms as compared to control (un-treated) worms
(Figure 4B.)
However, unlike our data in the BN-Page and SDS results (Figures 1 and 2) where a
dramatic difference was visible between exercised and food restricted worms, we were not able
to observe such a large difference between these conditions in the confocal data. Here, we
hypothesize that our sample preparation in PFA solution may have contributed to these results
i.e. that by placing worms into the solution, this allowed a window of time where all three groups
— control (un-treated), exercised, and food restricted — swam for a period of time. Given our
data in Figure 1, this may have resulted in smaller differences between exercised and food
restricted worms in particular.
While fixation using PFA solution has been effectively used in other research
(Laranjeiro et al. 2019), an alternative approach called for administration of levamisole — a
paralyzing agent which causes the fast contraction of muscle tissues — at 50 mM, in order to
immobilize and visualize worms (Goya et al. 2020). However, we were cautious to use this
chemical in the context of our experiments, as the contractions may reduce ⍺-synuclein protein
levels within the muscle cells. Instead, we utilized the PFA protocol where worms were fixed in
2% PFA solution directly after exercise and FR treatments (Laranjeiro et al. 2019).

30

Figure 3. Representative confocal images of ⍺-synuclein puncta in NL5901 worms. Puncta
(green) are shown after background subtraction was applied for A. control (un-treated) worms;
C. Ex worms; and E. FR worms, and with calculated Imaris surfaces (pink) for B. control (un-
treated) worms; D. Ex worms; F. FR worms. (N=3, n=~18-35 worms/replicate.)

31

Figure 4. Exercise and food restriction significantly reduce total puncta # per worm and average
puncta surface areas (SA’s.) A. Calculations show that Ex and FR worms have significantly
fewer puncta numbers than control (un-treated) worms (**p£0.0046, ****p<0.0001.) Ex worms
also have slightly more puncta, approaching significance (p£0.0563.) B. Confocal results also
show that Ex and FR worms have a significantly lower mean surface area (SA) than control (un-
treated) worms (****p<0.0001.) No difference in significance is seen between Ex and FR worms
(p=0.4578.) C. Confocal results show that FR worms have a significantly lower ratio of puncta
number to mean surface area (puncta #/mean SA) than control (un-treated) worms
(***p£0.0001) and Ex worms (*p£0.0217), indicating presence of larger puncta. Ex worms
appear to have some puncta with larger ratios than control (un-treated) and FR worms, indicating
the presence of smaller puncta, but this difference did not reach statistical significance
(p=0.4626.) All mean puncta and SA values were normalized to GFP intensity and control.

However, given our results in Figure 1, we were determined to observe if any differences
may be present between Ex and FR worms. Upon looking at these graphs more closely, we
noticed that while puncta numbers decreased slightly between exercised and food restricted
worms, mean surface area size stayed constant. Furthermore, in observations of individual
puncta data, we found that FR worms seemed to contain more puncta with larger surface areas
32
than Ex worms. Compounding these observations with our results in the western blot
experiments, we hypothesized that perhaps observing only the average SA values was
insufficient and that the average values may mask more subtle differences in puncta sizes. We
chose to explore further if the remaining puncta in FR worms are larger.
To accomplish this, we first calculated the ratio of puncta number to the mean surface
area of the puncta for each worm (puncta #/mean SA for each worm) (Figure 4C.) Interestingly,
we found that these ratios were significantly smaller for food restricted worms than for exercised
worms, indicating that the puncta which remain after 15-20 minutes of FR are larger than puncta
which remain after 15-20 minutes of Ex. Furthermore, the ratios seen in food restricted worms
are also significantly smaller than the ratios seen in control (un-treated) worms, which may
indicate a worsening of ⍺-synuclein aggregation i.e. formation of puncta which may be larger
than in control (un-treated) worms. Conversely, we also noticed that a number of exercised
worms contained ratios which are much larger than ratios present in control (un-treated) and food
restricted worms. Although the difference between control (un-treated) and Ex did not reach
statistical significance here, we hypothesized that this data could indicate that there are a number
of puncta in exercised worms which are considerably smaller than in the two other conditions
and may help to confirm the beneficial effects of exercise as observed by confocal microscopy.

ii. Observing size distribution of individual puncta per worm.

In order to further delineate the differences in puncta sizes across conditions as suggested
in Figure 4C, we collected data from each individual puncta from each individual worm (Figure
5.) In order to observe the distribution of puncta size among all worms, we graphed the results
using a heat map and found that exercised worms have a greater concentration of smaller puncta
33
(dark blue) than control (un-treated) or food restricted worms (Figure 5A.) Here, all values were
normalized to their respective controls and correspond to values from 0 to 3. We also graphed
the surface area values for all individual puncta for each worm and found that exercised worms
have a significantly smaller SA average than both control (un-treated) and food restricted worms
(Figure 5B.) Altogether, this data shows that by observing the surface area values of all
individual puncta per worm rather than the average surfaces areas of all puncta together per
worm, we see that there are indeed statistically significant differences between exercised and
food restricted worms where Ex worms have smaller puncta than FR worms. Although these
differences are again, not as dramatic as we observed consistently in BN-Page experiments
(Figure 1) and may be due to sample preparation as discussed above, these results ultimately
demonstrate that exercise and food restriction affect native ⍺-synuclein species differently in the
NL5901 worms.

34

Figure 5. Individual puncta show a statistically significant difference between exercised and
food restriction conditions. A. Heat map showing individual puncta for each worm across all
biological replicates. Exercise shows a higher concentration of smaller sized puncta shown in
dark blue (smaller than 2 units as seen in legend) than control (un-treated) and FR worms. B. Bar
graph showing all individual puncta SA’s for each condition. Exercise shows a significantly
lower SA average than food restriction conditions (****p<0.0001.) N=3, n=~18-35
worms/replicate.

In order to observe in further detail the differences between surface areas in control (un-
treated), exercised, and food restricted worms, we calculated the percentage of puncta in each
worm that were: 1) less than 5 um^2, 2) between 5 and 20 um^2, and 3) greater than 20 um^2.
These groups were chosen to reflect small, medium, and large protein aggregates and
corresponded loosely with the three groups we observed previously in Figure 1 i.e. 66-<480 kDa,
35
720 kDa, and well proteins, respectively. We also graphed our results in heat maps (Figure 6 A1-
C3) and quantified the differences in bar graphs (Figure 6 D-F.)
In our results, we found that exercised and food restricted worms have a significantly
higher percentage of puncta per worm that are smaller than 5 um^2 (Figure 6D.) Furthermore,
we found that there is a significantly smaller percentage of puncta that are between 5 and 20
um^2, as well as, greater than 20 um^2, in exercised and food restricted worms as compared to
control (un-treated) worms (Figure 6 E and F, respectively.) Altogether, these results may
indicate that larger puncta are broken down into smaller sizes during exercise and food
restriction, perhaps via heat shock proteins or changes in environmental conditions. In addition,
it may suggest that larger puncta are degraded preferentially over smaller puncta via protein
degradation or recycling pathways. While we did notice that food restricted worms have a
slightly lower percentage of worms with puncta smaller than 5 um^2, and a slightly greater
percentage of worms with puncta between 5 and 20 um^2 and larger than 20 um^2, these
differences did not reach significance. Because we did observe a significant difference between
Ex and FR worms in Figure 5, we hypothesize that the lack of a greater difference between Ex
and FR worms in Figure 6 may indicate the need for perhaps a different cutoff margin when
grouping the worms. This may lead to clearer differences between treatment groups.
36

37
Figure 6. Comparing analysis of three puncta size groups. Here, exercised and food restricted
worms have a greater percentage of puncta smaller than 5 uM^2 and a smaller percentage of
worms with puncta larger than 5 uM^2. A1-3. Heat map graphs show the distribution of surface
areas smaller than 5 um^2 for control (un-treated), Ex, and FR worms. B1-3. Heat map graphs
shown the distribution of surface areas greater than 5 um^2 and less than 20 um^2 for control
(un-treated), Ex, and FR worms. C1-3. Heat map graphs show the distribution of surface areas
greater than 20 um^2 for control (un-treated), Ex, and FR worms. D-F. Bar graphs show the
percentages of puncta that are less than 5 um^2, between 5 and 20 um^2, and greater than 20
um^2. Ex and FR worms contain a significantly higher percentage of puncta that are less than 5
um^2, and a significantly smaller percentage of puncta that are between 5 and 20 um^2, and
greater than 20 um^2, as compared to control (un-treated) worms (****p<0.0001.) (N=3, n=~18-
35 worms/replicate.)

IV. Summary: How a short treatment of exercise and food restriction in N5901 worms
effects changes in native human ⍺-synuclein protein species.

Overall, we observed that 15-20 minutes of swimming exercise on Day 1 of adulthood
causes a dramatic effect upon ⍺-synuclein protein quantity and species in BN-Page and SDS
page gels (Figures 1 and 2). Our data shows that after 15-20 minutes of swimming exercise,
worms appear to retain predominately a single band at ~90 kDa, which our preliminary results in
SDS gels have identified as the ⍺-synuclein-YFP monomer species. Our data is corroborated by
previous reports that have shown that dimeric and oligomeric species are significantly reduced in
a mouse model of diffuse Lewy body disorder after three months of voluntary running wheel
exercise (Zhou et al. 2017). Together, our results draw a parallel between the effects of exercise
in a mouse and C. elegans Parkinson’s model.
In support of our BN-Page data, confocal analysis showed that swimming exercise
significantly reduces total puncta number and, with respect to food restriction, reduced the mean
surface area of puncta when calculations were based on individual rather than average values
from worms (Figure 5). Food restriction also significantly reduced puncta number and average
surface area (Figure 4). In addition, both exercise and food restriction increased the percentage of
38
puncta smaller than 5 um^2, and reduced the percentage of puncta greater than 5 um^2 when
compared to control (un-treated) worms (Figure 6). This suggests that Ex and FR may
breakdown large aggregates forming smaller ones or preferentially target larger aggregates via
protein degradation pathways. Future experiments will involve exploring different confocal
and/or sample preparation methods which may help bring forth nuances delineating puncta
differences between exercised and food restricted worms.

39
Chapter 2. Observing effects of longer versus shorter exercise and food
restriction treatment times.

NL5901 worms experience a dramatic reduction in ⍺-synuclein protein species after just
15-20 minutes of swimming exercise. In order to better understand the changes in protein species
taking place as worms exercise for 15-20 minutes, we performed a timed experiment where we
analyzed worm samples from swimming bouts lasting 5, 10, and 15 minutes. In addition, we
aimed to address questions related to longer swimming bouts. Previous research utilized a 1.5
hour swimming bouts per exercise session in N2 worms, which led to improvements in a range
of healthspan parameters and protection in several models of tau and Huntingtin protein
aggregation (Laranjeiro et al. 2017; Laranjeiro et al. 2019). In order to observe how longer
periods of exercise may affect ⍺-synuclein protein species, we first tracked the swimming
patterns of worms over a period of 2 hours, where worm movements were recorded every 15
minutes for a period of 30 seconds. As a control for food restriction and temperature, we exposed
worms in parallel to conditions without food lasting 15-20 minutes, 1 hour and 2 hours, and ran
all samples in SDS gels in a preliminary experiment in order to roughly assess any changes
which these conditions may have induced. SDS gels are a commonly cited method for analyzing
⍺-synuclein protein species (Koo and Cho 2017; Shin et al. 2017; Zhou et al. 2017; Minakaki et
al. 2019). Once we established the swimming behavior of worms over a prolonged swimming
bout, we analyzed the changes in native ⍺-synuclein proteins for worms subjected to 1 and 2
hours of exercise and food restriction using BN-Page analysis.

40
I. Observing changes in native ⍺-synuclein species within 15 minutes of swimming exercise.

In order to observe the possible changes taking place in ⍺-synuclein protein species
during swimming exercise, we performed a timed experiment where worms were sampled
exactly at 5, 10, and 15 minutes (Figure 7). Here, our results show that just 5 minutes of
swimming exercise causes an almost significant reduction in the 66-<480 kDa range protein
species, underscoring the fast reductive effect of swimming exercise upon ⍺-synuclein species
(p=0.0597) (Figure 7A). At 10 minutes, there is a further gradual decrease in protein species
(Figure 7C), but a statistically significant effect is only observed at exactly 15 minutes of
swimming exercise.
In contrast to our previous results for 15-20 minutes of exercise (Figure 1), where there is
a significant reduction in 720 kDa and well proteins, here we do not see a significant reduction in
either of these species within the 15-minute swimming time, although there is a trend towards
significance for well proteins at exactly 15 minutes of exercise (p=0.0651) (Figure 7C and D).
This data highlights the possibly faster reductive effect of exercise on protein species within the
66-<480 kDa range and that, in order to observe a significant reduction in higher molecular
weight species, worms require longer periods of swim time i.e. 15-20 minutes versus 15 minutes
or less.

41

Figure 7. Exercise shows a progressive reduction in protein quantity every 5 minutes, and the
emergence of a single predominant band at 15 minutes of exercise. A. Protein densitometry
analysis of 66-<480 kDa range proteins shows that 5 minutes of exercise shows a trend towards a
42
significant reduction in protein quantity (p=0.0597), followed by 10 minutes of exercise
(p=0.0644.) However, a significant decrease is only observed at exactly 15 minutes of exercise
(*p≤0.0124.) B. A representative blot shows the gradual reduction in ⍺-synuclein protein species
from 0 to 15 minutes of exercise (bracketed lanes are one sample split into two lanes.) The pink
arrow indicates the emergence of a predominant single band over time. C. Densitometry analysis
of 720 kDa proteins shows no significant differences from 0 minutes to 15 minutes of exercise.
D. Densitometry analysis of well proteins shows a gradual reduction with time but with only a
trend towards significance at exactly 15 minutes of exercise (p=0.06551.) (N=3, n=25
worms/replicate.)

In addition, it is interesting to note that while these shorter exercise intervals also show a
reduction of proteins in the 66-<480 kDa range similar to the degree observed after 15-20
minutes of food restriction, the pattern here is different from that which is observed in food
restricted worms — exercised worms show proteins are concentrated in certain regions, such as
above or below ~146 kDa and at ~90 kDa after 5 minutes of exercise, whereas food restricted
worms mostly show a range of proteins, and rarely show areas where proteins concentrate.
Unfortunately, the lack of a consistent pattern in each biological replicate did not allow
for the exact determination of the predominant species present at each 5-minute interval. We
hypothesize that this variability may be due to stochasticity where worms may intrinsically
contain different species that vary between biological replicates. Despite this possibility, we were
able to observe in all replicates that while 5 and 10 minutes showed a range of proteins or several
distinct bands, exactly 15 minutes of exercise consistently resulted in a prominent single band. In
future experiments, we will aim to further track the gradual changes taking place in ⍺-synuclein
species within 15 minutes in order to better understand how exercise affects native ⍺-synuclein
species in a stepwise manner i.e. which species are reduced first, etc.

II. Timed experiment observing NL5901 thrashing speeds.

43
Before beginning experiments that involved prolonged periods of swimming for the
NL5901 worms, we first aimed to quantitate their swimming behavior by assessing changes in
thrashing rates with time. We were principally concerned that the presence of ⍺-synuclein
protein species within the worm muscles may require adjustments to the extended exercise
protocol previously established for N2 worms (Laranjeiro et al. 2017; Laranjeiro et al. 2019). It
was previously determined that N2 worms experience exhaustion from swimming exercise at
approximately 30 minutes to 1 hour after they begin swimming, further indicating that swimming
is a strenuous form of activity constituting exercise (Laranjeiro et al. 2017). At this time, worms
begin to enter into a “lag phase,” which is characterized by brief periods of rest i.e. where worms
pause body bend movement on either side of their body (Schuch et al. 2020). We have observed
that this pause, which in NL5901 worms occurs sometimes as a brief hesitation or other times
lasts a few seconds, is usually followed by a vigorous return to swimming. Accumulation of such
pauses during the scoring period however, contribute to the overall reduction in average
thrashing rates.
In order to carefully monitor the swimming behavior of NL5901 worms, worms were left
undisturbed under a recording microscope for a period of 2 hours. Swimming patterns were
recorded every 15 minutes for 30 second intervals, and thrashing rates were determined from the
recorded video at a later time. Here, we observed that worms maintain a relatively even thrashing
speed for about 45 minutes to 1 hour (Figure 8A). However, at around the 1-hour time mark, the
thrashing speed becomes significantly slower. This behavior supports previous observations
made in N2 worms, where worms exhibited signs of exhaustion at this time (Laranjeiro et al.
2017). Furthermore, this data suggests that aside from the slower thrashing rates reported in
NL5901 as compared to N2 worms (Anand et al. 2020), NL5901 worms respond similarly to
44
exercise. Interestingly, we noticed that the thrashing rates go up and down in a cyclic pattern
approximately every 15 minutes between 1 and 2 hours of swimming exercise. We hypothesize
that these preliminary observations suggest entry of worms into the “lag phase.”
At the conclusion of the 2-hour experiment, exercising worms were collected for SDS
page gel analysis (Figure 8 B-D.) Our main intention with this experiment was to observe if any
abnormal changes may have occurred to ⍺-synuclein proteins as a result of environmental
factors, such as the increased temperature of the microscope room, which may inform our results
from the thrashing assay. Here, we analyzed samples from control (un-treated), food restricted
(15-20 minutes, 1 hour, and 2 hours) and worms exercised for 2 hours (which are the same
worms from the thrashing experiment.)

45

46
Figure 8. Preliminary results from a timed experiment show the quantification of thrashing rates
in NL5901 worms at 15-minute intervals as well as corresponding samples from SDS gel. A.
NL5901 worms appear to present a varying pattern of thrashing speeds from 1 hour to 2 hours,
suggesting entry into the “lag phase,” a phenomenon also observed in wild type worms. 1 hour, 1
hour and 15 minutes, and 1 hour and 45 minutes show significantly lower thrashing rates than at
15-20 minutes (**p≤0.0015, *p≤0.0273, and**p≤0.0018, respectively.) B. SDS page gel
showing the protein species present at the monomeric level and below for worms subjected to
food restriction for 15-20 minutes, 1 hour, and 2 hours, as well 2 hours of exercise from worms
subjected to 2 hours of thrashing assay in (A) C. Enhanced SDS page gel showing higher
molecular weight proteins where dimers appeared to be increased in worms exercised for 2
hours. D. Quantification of monomeric, dimeric, and hexameric protein species from (C)
showing that exercised worms have approximately a 4 to 5-fold induction of dimeric species as
compared to control (un-treated) and 2 hours of food restriction, respectively. (N=1, n=~50 in A.;
n=~25 worms in B-D.)

In our results, we see that all time points for food restriction exhibited a decrease in
dimeric and hexameric species as compared to control (un-treated) worms (Figure 8 B-D.)
Interestingly, we observed that while 2 hours of exercise reduced the hexameric ⍺-synuclein
protein species as compared to control (un-treated) worms, there was an increase in dimeric
species almost 4-fold as compared to control (un-treated) worms, and 4 to 5-fold as compared to
worms exposed to food restriction for 2 hours. While these experiments are preliminary, they
indicate an interesting effect. In addition, we have also noted similar results in separate
experiments where worms were exercised for 3 hours and compared to non-exercising worms on
bacterial (not food restricted) plates (data not shown.) In particular, we observed a greater
presence of oligomeric species, such as dimers, in exercised versus non-exercised worms (data
not shown). While many factors may have contributed to our results, future experiments will
explore the possibility that longer periods of exercise exposure may yield an increased presence
of dimers, perhaps due to post-translational modifications by ROS, which may be increased
under exercise conditions. As previously mentioned, while ROS expression can be detrimental,
47
research has observed their ability to promote formation of protective oligomeric ⍺-synuclein
species (Wördehoff et al. 2017).
Altogether, our results show that NL5901 worms appear to behave similarly to N2 worms
as previous reported, likely entering the “lag phase” at approximately the same time i.e. around
the 1-hour time point. In addition, analysis of samples from the exercised and food restricted
worms in SDS page gels showed that both sets of conditions exerted a reductive effect upon ⍺-
synuclein species and, that perhaps, a prolonged period of exercise may yield formation of
dimeric ⍺-synuclein.

III. Observing effects of longer exercise and food restriction treatments upon native human
⍺-synuclein species.

After understanding the behavioral patterns of NL5901 worms subjected to longer bouts
of swimming exercise, we began experiments analyzing their protein profiles using BN-Page
gels. Here, we subjected worms to 1 and 2 hours of swimming exercise and compared the results
to 15-20 minutes of exercise (Figure 9). Here, we see that 1 and 2 hours of exercise do not
further decrease the quantity of 66-<480 kDa and 720 kDa proteins, while there are some
fluctuations in well proteins (Figure 9 D1-D3). Importantly, 1 and 2 hours of exercise maintain
the same protein profile in the 66-<480 kDa range as observed after 15-20 minutes of exercise
(Figure 9 A-C).
Interestingly, we found an almost significant increase in well proteins in worms that
exercised for 1 hour (p=0.0651) and sometimes a lower presence of well proteins after 2 hours
(Figure 9 D3). However, these differences were not statistically significant. Previously, we
showed that NL5901 worms seem to enter the “lag phase” after approximately 1 hour of
48
swimming (Figure 8). Together from data in Figures 8 and 9, this indicates that entry into the
“lag phase” for NL5901 worms may be associated with an increase in well protein levels and
may explain the slower thrashing rate. It may also suggest that a prolonged period of exercise
may promote ⍺-synuclein aggregation via ROS production as mentioned in Section II. At 2
hours, however, well protein levels return to quantities observed at 15-20 minutes, and
interestingly, this corresponds to an increase in thrashing rate (Figure 9 D3 and Figure 8,
respectively). It is possible that exercised induced ROS can reversibly react with proteins at
methionine residues, and perhaps this may explain the increase in protein quantity at 1 hour, and
then the subsequent decrease at 2 hours (Filomeni et al. 2015). In addition, it may perhaps be
possible that mechanisms of protein clearance are cyclical, fluctuating throughout the exercise
interval, and perhaps resulting in altered levels of protein aggregation. Future experiments will
explore these possibilities further and attempt to track temporal changes in protein levels and
mechanistic action.

49

Figure 9. Effects of 1 and 2 hours of exercise and food restriction. 1 and 2 hours of exercise and
food restriction do not further reduce ⍺-synuclein protein quantity or change protein profiles
within the 66-<480 kDa range. There is also no further change in 720 kDa proteins, but 2 hours
of FR significantly reduces well proteins. A. Representative BN-Page western blot showing 15-
20 minutes and 1-hour treatments of exercise or food restriction. B. An enlarged image
highlights the 66-<480 kDa region indicating the presence of bands not visible in A. C.
Representative BN-Page western blot shows the effect of 2 hours of exercise or food restriction
treatments as compared to 15-20 minutes. D1-D3. Protein densitometry analysis of exercised
worms shows that 66-<480 kDa (D1), 720 kDa (D2), and well proteins (D3) do not further
reduce protein species as compared to 15-20 minutes of exercise. However, there is an almost
significant increase in well proteins due to 1 hour of exercise (p=0.0651.) E1-E3. Protein
densitometry analysis shows that 1 and 2 hours of food restriction do not show a significant
decrease in 66-<480 kDa (E1) nor 720 kDa (E2) proteins as compared to 15-20 minutes. In
50
contrast, 2 hours of food restriction yields a significant decrease in well proteins as compared to
15-20 minutes (E3) (*p≤0.0487.) This highlights the possible differential effect of food
restriction on lower verses higher molecular ⍺-synuclein protein species. (N=4, n=25
worms/replicate.)

In addition to results in exercising worms, we observed no significant changes in food
restricted worms in the 66-<480 kDa and 720 kDa protein species as compared to 15-20 minutes
of FR (Figure 9 E1 and E2). In addition, these longer periods of food restriction do not further
alter the protein profile in the 66-<480 kDa range as compared to 15-20 minutes — this region
continues to show a range of proteins, sometimes with a more or less concentrated band. This is
in contrast to protein in exercised worms, which exhibit only a crisp, single band. In addition, in
a separate experiment (data not shown), we observed that exposing worms to 24 hours of food
restriction did not ultimately lead to depletion of ⍺-synuclein proteins as compared to worms that
were exposed to food restriction for 15-20 minutes and placed back onto bacterial plates.
Altogether, these results suggest that just as longer bouts of exercise do not further reduce
proteins between 66-<480 kDa, longer periods of food restriction also reach a maximum extent
of protein depletion after 15-20 minutes of treatment. This suggests that increasing the time of
food restriction will not further reduce protein levels, nor change the protein profile, in such a
way that it becomes equivalent to the results we have observed in swimming exercise. This data
further highlights that exercise and food restriction appear to affect ⍺-synuclein species
differently and cannot be interconverted.
In contrast to the effects observed upon 66-<480 kDa proteins in response to food
restriction, we noticed that exposing worms for 2 hours to FR results in a significant decrease in
well protein when compared to 15-20 minutes of FR. It is interesting to note that, while these
larger protein species are reduced after 2 hours, the 66-<480 kDa range proteins remain at the
51
same level as at 15-20 minutes, highlighting a possibly differential temporal effect of food
restriction on smaller versus larger molecular weight ⍺-synuclein protein species.

IV. Summary: What both shorter and longer bouts of exercise and food restriction can tell
us about the differential effects of these treatments upon native human ⍺-synuclein protein
species

Overall, our data shows that within 15 minutes, swimming exercise shows a gradual
reduction in protein species, beginning as early as 5 minutes. Unlike in the effects of 15-20
minutes of food restriction, which exhibits a similar reduction in proteins as a result of exercise,
this gradual shift in Ex worms results in several distinct areas of protein concentrations within
the 66-<480 kDa range. In addition, we only observe a significant reduction exactly at 15
minutes of exercise in the 66-<480 kDa range, but do not observe a significant decrease of 720
kDa and well proteins.
In observing the effects of swimming exercise lasting up to 2 hours, we found that our
data shows that, aside from slower thrashing rates, NL5901 worms behave in a similar manner to
N2 worms, slowing down at approximately 1 hour after exercise is initiated and likely entering
the lag phase at this time. In addition, our data suggests that perhaps exposing worms to longer
periods of exercise, such as for 1 hour, may lead to increased well proteins, indicating either a
build-up of protein due to aggregation — perhaps as a result of reversible reactions with ROS —
or a temporary decrease in protein degradation pathways.
In addition, we found that 1 and 2 hours of exercise and food restriction do not further
reduce the protein quantity, nor the protein profile, of the 66-<480 kDa region, suggesting that
15-20 minutes of exercise and food restriction yields the maximum reduction in these protein
species. Conversely, we observed a significant decrease in well proteins after 2 hours of food
52
restriction, indicating a differential effect of FR upon smaller versus larger molecular weight
species. Future experiments will focus on determining if 1 and 2 hours of exercise results in a
cyclic effect upon protein aggregates, possibly mediated by ROS, and/or changes in protein
degradation pathways.

53
Chapter 3. Observing the downstream effects of exercise or food restriction
upon NL5901 worms.

In order to determine the longer-term downstream effects of exercise and food restriction
upon NL5901 worms, we assessed the thrashing abilities of all worms at two time points — one
time point observed the thrashing ability of worms close to their state while on bacterial plates,
while the other time point assessed the thrashing ability of the same worms after a bout of
swimming exercise. Here, we noticed that food restricted worms behaved altogether differently
than either control (un-treated) or previously-exercised worms, suggesting a very interesting
differential downstream effect of food restriction upon ⍺-synuclein protein species. We further
assessed changes in ⍺-synuclein protein species 1 hour and 24 hours after exercise or food
restriction treatments. Here, we observed significant differences between exercised and food
restricted worms at both time points. Our analysis of ⍺-synuclein protein species at 24 hours
post-treatment specifically, appears to further explain our results from the thrashing assays.

I. Analyzing thrashing behavior of previously-exercised or food-restricted worms.

Thrashing assays are commonly used as a readout to quantify worm neuromuscular
function. In previously established protocols, thrashing assays have involved the placement of
worms into a physiological solution i.e. S-basal, allowing worms to equilibrate to the solution for
30 seconds, and quantifying the rate at which worms thrash in the consecutive 30 second
interval. A period of equilibration is required to allow worms to adjust to their new environment,
such as the different osmotic pressure presented by submersion in water. The total time,
therefore, for one thrashing assay per worm, is 1 minute. However, we observed in previous
experiments (discussed in Chapter 4) that NL5901 worms appear to require a longer period of
54
equilibration than N2 worms, often appearing very slow or less mobile initially, and gaining
greater speed after 1 minute. We also observed that NL5901 thrashing rates can vary
considerably from worm to worm in one biological replicate. As has been previously observed,
NL5901 worms have average thrashing rates that can range from approximately 10 to 35
thrashes per 30 seconds (Anand et al. 2020). Our results support these observations. However,
we have found that a number of worms in each condition can exhibit higher thrashing rates,
reaching values between 40 and 50 thrashes per 30 seconds, which are akin to rates observed in
N2 worms. This considerable variability suggests that the degree of ⍺-synuclein aggregation may
be different from worm to worm, something which we have observed in protein profiles between
biological replicates as well. In order to decrease the effect of this variability, we altered the
thrashing assay protocol by utilizing a recording microscope, where worms were recorded on the
day of the experiment, but scored at a later time. This technique has allowed us to have a higher
throughput — a range from 25 to 50 worms recorded at one time — while same day experiments
usually only allow for approximately 15 worms per condition in one biological experiment. This
modification has also allowed us to observe all worms in the sample at exactly the same time and
under the same conditions, reducing further sources of variability.
As previously discussed in Chapter 2, we determined that the 66-<480 kDa ⍺-synuclein
protein species begin to decrease after 5 minutes of swimming exercise. In order to avoid this
effect yet still provide an appropriate amount of time for worms to equilibrate to the liquid
solution, we chose to assay worms at 3 minutes, recording their swimming patterns here during a
30 second interval. At this time, the full effect of exercise should not significantly affect ⍺-
synuclein protein levels and worms are likely to possess protein levels at similar levels to their
state while on bacterial plates.
55
In addition to experiments at 3 minutes, we also observed how worm thrashing speeds
may change temporally by leaving worms swimming undisturbed under the recording
microscope up to 20 minutes. In this way, we were able to observe the effect of swimming
exercise on ⍺-synuclein protein species and/or health of the worms in vivo. Overall, quantifying
worm thrashing rates and determining the changes between 3 and 20 minutes in the control,
exercised, and food restricted conditions, has allowed us to observe the differential effects of
exercise upon worms in each condition.
As the following subsections will show, worms were assayed on two days — Day 2 and
Day 4 i.e. 24 hours and 96 hours after exercise or food restriction conditions — at the two time
points mentioned previously, 3 minutes and 20 minutes. We attempted, but were not fully able to
complete, experiments for worms on Day 9 due to increased bagging observed in older NL5901
worms — a common problem we have encountered in our work with this strain. Although use of
5- fluoro-2′-deoxyuridine (FUdR) would have alleviated this problem, we chose to initially avoid
its use over concerns about potential effects and/or possible interferences in exercise mechanisms
— FUdR was shown to increase the lifespan of gas-1 mutants (reduced function of
mitochondrial Complex I protein) (Van Raamsdonk and Hekimi 2011). Despite these
considerations, this is still a viable option for future experiments in order to observe the effects
of Day 1 exercise and food restriction on NL5901 worms at a greater age.

i. Thrashing assay analysis at 24 hours (Day 2) post-exercise or food restriction.

As previously described, Day 1 worms were exposed to exercise or food restriction
conditions for 15-20 minutes. Afterwards, worms were placed back onto bacterial plates and
subjected to a thrashing assay at 24 hours (Day 2) post-treatment. Figure 10 shows that
56
previously exercised worms have a statistically significant higher thrashing rate, approximately
16 thrashes per 30 seconds, than control (un-treated) and food restricted worms which have
thrashing rates of approximately 12 and 10 thrashes per 30 seconds, respectively. There is no
statistically significant difference in thrashing rates between control (un-treated) and food
restricted worms. Altogether, the data in Figure 10 shows that worms exercised on Day 1 retain
the benefit of exercise for 24 hours, while food restricted worms, which also experience a
different, yet significant reduction in 66-<480 kDa and well proteins (Figure 1), exhibit the same,
if not slightly slower on average thrashing rates as control (un-treated) worms. As the thrashing
ability of NL5901 worms depends upon the aggregation of ⍺-synuclein protein species, this data
suggests that the degree of aggregation is likely less in previously-exercised worms than in either
the control (un-treated) or food restricted conditions 24 hours post-treatment.

57
Figure 10. Thrashing assay results on Day 2 (24 hours post-treatment) at the 3 minutes. Results
show that on Day 2, worms that previously exercised for 15-20 minutes on Day 1 have a
significantly higher thrashing speed at 3 minutes compared with control (un-treated) (*p£0.0250)
or food restricted (FR) worms (
##
p£0.0027). (N=3, n=21-36 worms per replicate.)

In order to confirm the protective effect of a single bout of exercise on worms in vivo,
worms were allowed to remain swimming under the recording microscope until the 20-minute
mark, which corresponds to a full bout of swimming exercise. Here, we observed that control
(un-treated) worms had a statistically significant improvement in their thrashing speed to
approximately 19 thrashes per 30 seconds, attaining a speed which is similar to previously
exercised worms (approximately 18 thrashes per 30 seconds) (Figure 11.) While this is a
significant improvement for control (un-treated) worms from their speed at 3 minutes (Figure
10), food restricted worms were not able to improve their thrashing speed over the 20-minute
exercise period, and maintained the same average 10 thrashes per 30 seconds at the end of 20
minutes. This data indicates that food restriction on Day 1 may have negative effects on either ⍺-
synuclein aggregation and/or the health of the worms after 24 hours.

58

Figure 11. Thrashing assay results after 20 minutes of exercise on Day 2 (24 hours post-
treatment.) Results show that control (un-treated) worms have attained a higher thrashing rate,
matching that of Ex worms and are significantly faster than FR worms (****p<0.0001), Ex
worms maintain approximately the same thrashing speed as at 3 minutes, which is also
significantly higher than for FR worms (
###
p£0.0003). FR worms do not appear to see an
increase in thrashing speed from 3 minutes to 20 minutes. (N=3, n=21-36 worms per replicate.)

ii. Thrashing assay analysis at 96 hours (Day 4) after exercise or food restriction.

Thrashing assays were also performed on worms at 96 hours (Day 4) post-exercise and
food restriction treatments. Unlike on Day 2, the worms did not exhibit any differences between
the three conditions, albeit all displayed lower thrashing rates than on Day 2 (Figure 12A.) The
phenomenon of lower thrashing rates with age has previously been observed in N2 worms as
well (Hahm et al. 2015). After 20 minutes, however, previously exercised worms achieved a
59
significantly higher thrashing speed than control (un-treated) worms and a trend towards a
significantly higher speed than food restricted worms (p=0.0673) (Figure 12B.) In addition, after
20 minutes, exercised worms achieved a thrashing speed similar to that of control (un-treated)
worms on Day 2, suggesting that exercise exerts a restorative effect upon older NL5901 worms.
We also noticed that, unlike on Day 2, food restricted worms were able to improve their
thrashing speed between 3 minutes and 20 minutes, indicating that any negative effect which
food restriction may have had on worms 24 hours post FR, was reduced by Day 4.

Figure 12. Thrashing assay results on Day 4 (96 hours post-treatment.) A. Results demonstrate
that at 3 minutes, all three treatment groups (control (un-treated), Ex, and FR) display
approximately the same lower thrashing speed as compared to Day 2 results. B. Thrashing assay
results on Day 4 at 20 minutes show that Ex worms have a significantly higher thrashing rate
than control (un-treated) worms (*p£0.0371) and an almost significant trend towards a higher
thrashing rate compared with FR worms (p=0.0673). All worms show an improvement in
60
thrashing rate compared to their speed at 3 minutes; Ex worms show an average thrashing speed
similar to that of control (un-treated) worms on Day 2. (N=3, n=22-30 worms per replicate.)

Altogether, these results highlight that a single 15-20 minute bout of exercise on Day 1 is
able to improve the outcome of worm health and/or ⍺-synuclein aggregation in worms four days
later. Although we were not able to complete experiments for Day 9 worms, we hypothesize that
given our results on Day 4, it may be possible that the ⍺-synuclein protein levels may remain
lower, and/or the fitness level of previously exercised worms will still remain at a higher level,
than in control (un-treated) worms after 20 minutes of exercise treatment.

II. Analysis of ⍺-synuclein protein species 1 hour after exercise or food restriction
treatments.

Upon recovery from exercise, organisms utilize carbohydrates in order to restore protein
levels (Brooks et al.). OP50 bacteria serve as a source of proteins, carbohydrates, and lipids for
C. elegans worms (Watts and Ristow 2017). In order to determine if ⍺-synuclein protein species
will be restored downstream after swimming exercise, worms were placed back onto bacterial
plates for 1 hour i.e. re-fed (RF). Interestingly, we observed a very strong average increase in
protein concentrations in this time frame in all three species analyzed: proteins located in the 66-
<480 kDa range showed an almost 6-fold increase (Figure 13A); 720 kDa proteins showed an
almost 27-fold increase (Figure 13C); well proteins showed a 13-fold increase (Figure 13D.)
This strong increase in protein after only 1 hour of re-feeding may suggest a reduction in any
mechanisms responsible for decreasing protein aggregates, such as autophagy, during exercise or
food restriction treatments.
61
While food-restricted worms also experienced a significant increase in the 66-<480 kDa
range proteins and in well proteins equal to about 6-fold (Figures 13 B and D), they did not
exhibit a significant increase in 720 kDa proteins (p=0.1285) (Figure 13 C). As it was previously
observed in Figure 1 that food restriction did not significantly decrease protein quantity to the
same degree as exercise, these fold-increases align with our previous results. Interestingly, it
appears that, overall, exercised worms gained slightly more protein within 1 hour of re-feeding
than food restricted worms, especially in 720 kDa and well proteins. However, these differences
were not statistically significant (p=0.4278 and p=0.2312, respectively).
62

Figure 13. Re-feeding (RF) worms for 1 hour post 15-20 minutes of exercise or food restriction
causes a significant increase in ⍺-synuclein protein species. A. Protein densitometry analysis of
66-<480 kDa range proteins shows that 1 hour of RF post exercise and food restriction causes an
63
almost 6-fold increase in protein quantity (
&
p≤0.0242,
#
p≤0.0448, respectively.) B.
Representative BN-Page western blot shows the dramatic increase in protein species after just 1
hour of RF. C. Densitometry analysis of 720 kDa proteins shows that while exercised worms
experience a significant ~27-fold increase in protein levels (
&&
p≤0.0083), food restricted worms
do not experience a significant increase in protein levels (p=0.1285.) D. Densitometry analysis of
well proteins shows that exercised worms experience about a 13-fold significant increase in
protein quantity (
&
p≤0.0497.) Similarly, food restricted worms show a significant ~6-fold
increase in well proteins at 1 hour of RF (
#
p≤0.0482.) (N=3, n=~25 worms/replicate.)

Interestingly, after both exercise and food restriction treatments, worms had an increase
in the 66-<480 kDa proteins to values that were, on average, below values for control (un-
treated) worms (Figure 13A). Conversely, both Ex + 1 hr. RF and FR + 1 hr. RF worms showed
an increase of 720 kDa and well protein species to values that were on average higher than
control (un-treated) worms, suggesting that higher molecular weight species are restored more
quickly than lower molecular weight species (Figure 13B and C.) While these differences
between treated and control (un-treated) worms were not statistically different, this data may
provide us with some information about the progression of protein aggregate formation in
NL5901 worms after exercise and food restriction treatments are administered. Currently, we do
not know exactly how protein aggregates form in NL5901 worms, although previous research
has shown that aggregates are present from the L1 stage and continue growing as the worms age
to adulthood (Goya et al. 2020). However, observations of ⍺-synuclein aggregate formation in
general tend to show a progression from smaller to larger aggregates (Lashuel et al. 2013).
Therefore, our data in Figure 13 may suggest that, at least initially, formation of higher
molecular weight species aggregates may be biochemically favorable over lower molecular
weight species until equilibrium is achieved. Future time course experiments may help answer
these questions by investigating the progression of aggregate formation between 15-20 minutes
to 1-hour post-treatment.
64
Altogether, this data demonstrates that there is a dramatic increase in ⍺-synuclein protein
quantity in exercised worms across all species observed and, in food restricted worms, an
increase in all proteins except 720 kDa proteins. In addition, while not significant, exercised
worms appear to have a greater quantity of proteins for all three species analyzed when
compared to food restricted worms. This may suggest that post exercise mechanisms may be
driving an initially greater increase in ⍺-synuclein protein restoration upon re-feeding than after
food restriction conditions.

III. Analysis of ⍺-synuclein protein species 24 hours post exercise or food restriction
treatments.

As seen in earlier in thrashing results in Figures 10 and 11, while control (un-treated)
worms experienced an increase in their thrashing speed after 20 minutes of exercise, food
restricted worms maintained a consistently low thrashing speed throughout. Here, we analyzed
BN-Page westerns where we see that worms kept on bacterial plates for 24 hours (until Day 2)
post-exercise contain a significantly smaller quantity of higher molecular weight species (720
kDa proteins and well proteins) than food-restricted worms (Figure 14 C and D.) This data
further corroborates results observed in confocal analyses in Chapter 1, where FR worms showed
larger ⍺-synuclein-YFP puncta than Ex worms (Figure 5.) We hypothesize that these results may
help explain the lower thrashing rates of food restricted worms on Day 2 at 3 minutes as
compared to exercised worms (Figure 10). This data also suggests that higher molecular weight
species are more likely to affect the thrashing ability of worms than lower molecular weight
species. Due to their greater size and their location in muscle cells, it seems logical that these
protein species may physically pose a greater hindrance to C. elegans swimming patterns.
65

Figure 14. Re-feeding (RF) worms for 24 hours after 15-20 minutes of exercise or food
restriction. Here, analysis shows a significantly smaller quantity of higher molecular weight ⍺-
66
synuclein protein species in exercised versus food restricted worms. A. Densitometry analysis of
66-<480 kDa range proteins shows that while a difference in protein levels is present at 24 hours
of RF, this difference is not significant among the biological replicates (p=0.2124.) B. A
representative BN-Page western blot highlights the greater presence of 720 kDa and well
proteins in food restricted versus exercised worms. C. Densitometry analysis shows that
exercised worms show a significantly smaller quantity of 720 kDa proteins at 24 hours of RF as
compared to food restricted worms (*p≤0.0444.) D. Densitometry analysis of well proteins
shows that exercised worms contain a significantly smaller quantity of well proteins at 24 hours
of RF as compared to food restricted worms (*p≤0.0294.) (N=3, n=~25 worms per replicate.)

In addition, as we see in Figure 11, these results suggest that 15-20 minutes of food
restriction impacts not only the quantity of proteins in Day 2 FR worms, significantly increasing
levels past those seen in exercised worms, but also hinders the clearance of these proteins due to
mechanisms likely upregulated during exercise. This suggests a possibly important relationship
between low nutrient availability and proteotoxicity and begs the question whether food
restriction may result in detrimental post-translational modifications to ⍺-synuclein species.
Similar to mammals, C. elegans worms utilize carbohydrates to produce glucose, an
important source of energy (Watts and Ristow 2017). However, it has been shown that while
treatment with excess glucose reduces C. elegans longevity, inhibiting glucose transporters or
treating N2 worms with glucose inhibitors increases longevity through generation of ROS
(Heidler et al. 2010; Schmeisser et al. 2013). Here, these effects have been shown to be mediated
through DAF-16 and SIR-2.1 or neuronal ROS signaling.
Interestingly, however, it has been shown that with regard to the effect of glucose on
proteotoxicity, excess glucose alleviates proteotoxicity in different C. elegans models of
Huntington’s, ALS, and Alzheimer’s (Tauffenberger et al. 2012). Here, it was also shown that
dietary restriction does not alleviate aggregation of proteins in these models, although here, DR
was performed chronically — from Day 1 to Day 12. Glucose deprivation has also been shown
to increase ⍺-synuclein aggregation in human dopaminergic SHSY5Y cells where the authors
67
implicated ROS production (Bellucci et al. 2008). Altogether, this data suggests that dietary
restriction may have a detrimental effect upon protein aggregation.
These results also introduce another question: because ROS levels increase in response to
exercise and caloric restriction, why do exercised worms not experience a similar worsening in
⍺-synuclein proteotoxicity? As discussed previously, ROS can have a divergent effect upon
different ⍺-synuclein proteins species (Wördehoff et al. 2017). Namely, ⍺-synuclein interaction
with ROS can result in both protective oxidized species that inhibit aggregation and growth —
when ⍺-synuclein monomers or dimers react with ROS — or detrimental species which promote
aggregation — when ⍺-synuclein fibrils react with ROS. In previous results, we observed that
the quantity and species remaining after exercise and food restriction are different (Figure 1).
While exercised worms predominantly contain the monomeric species immediately after
treatment, food restricted worms contain most species present in control (un-treated) worms,
except in a smaller quantity. We hypothesize that under exercise conditions, the predominantly
monomeric ⍺-synuclein species interact with ROS and produce protective species which resist
growth and aggregation. Conversely, we propose that under food restriction, the remaining ⍺-
synuclein oligomeric species react with ROS and produce species that propagate aggregation.
Another possibility explaining the positive effects of exercise may have to do with an
improvement in energy production, which may occur through the mobilization of glucose as well
as the generation of lactate. It has been previously shown that swimming exercise upregulates the
lactate dehydrogenase (ldh-1) gene in C. elegans and our preliminary results have shown
upregulation of ldh-1 after 15-20 minutes of exercise (data not shown) (Laranjeiro et al. 2017).
Despite earlier research which may have misconstrued lactic acid to be a waste product of
exercise, more recent work has shown that it is an important energetic molecule and
68
supplementation has been suggested to improve exercise performance, as well as to provide
greater rehabilitation after traumatic brain injury (Patet et al. 2016; Brooks 2020; Brooks et al.).
Perhaps exercised worms do not experience the same worsening of ⍺-synuclein protein
aggregation over time as food-restricted worms because of the improved energy production from
glucose and/or increased lactate levels.

IV. Summary: Observing downstream effects of exercise and food restriction.

Overall, our results demonstrate that 15-20 minutes of swimming exercise induces
downstream protective effects upon NL5901 worms, where worms exhibit faster thrashing rates
than control and food restricted worms on Days 2 and 4. Interestingly, while we observed that
while 15-20 minutes of food restriction appears to have a short-term ameliorative effect upon ⍺-
synuclein protein quantity immediately after treatment, it seems to have a detrimental effect after
24 hours of re-feeding conditions. Here, worms have a significantly higher quantity of 720 kDa
and well proteins than exercised worms, and are not able to experience an improvement in
thrashing rate after 20 minutes of exercise when compared to control (un-treated) worms. Here,
we hypothesize that ROS, which are induced under both conditions, may play a role in the
divergent effects between exercise and food restriction, leading to protective and pro-aggregative
⍺-synuclein species, respectively. This data may further suggest that previously food restricted
worms contain ⍺-synuclein protein species that are not amenable to the ameliorative mechanisms
induced under exercise conditions.

69
Chapter 4. Investigating the effects of pharmacological interventions on C.
elegans NL5901 and GMC101 worms.

Our results so far have shown that a short 15-20 minute session of exercise or food
restriction results in significant changes to native human ⍺-synuclein species within the NL5901
worm. Furthermore, despite the possibility that exercised worms experience a degree of food
restriction due to lower pharyngeal pumping as discussed in Chapter 3, we noticed that these
treatments resulted in different effects upon the ⍺-synuclein protein profiles and, while exercise
appears to have a protective effect, food restriction exhibits some detrimental aspects. Previous
research has pointed to the roles of the proteasome and autophagy as mediators of protein
degradation in response to exercise and caloric restriction (Koo and Cho 2017; Sampaio-
Marques et al. 2018; Chen et al. 2019; Minakaki et al. 2019). In particular, we were very
interested in the possible role of ROS. Here, we utilized the proteasomal inhibitor,
carbobenzoxy-Leu-Leu-leucinal (MG132) and the anti-oxidant, N-acetyl-l-cysteine (NAC) in
order to assess the possible role of the proteasome or autophagy (as mediated through ROS),
respectively. In addition, we also performed several preliminary experiments with a novel natural
product HIF-1⍺ inducer, Diphyllin (DP), and present here results which demonstrate its capacity
for clearance of ⍺-synuclein species.

I. Investigating the role of protein degradation pathways under exercise and food
restriction conditions.

Our previous data has shown that there are differences in the effects of exercise and food
restriction upon the three groups of ⍺-synuclein species analyzed. In particular, we have seen
that 15-20 minutes of exercise significantly reduces proteins in the 66-<480 kDa range to a
single, monomeric band at ~90 kDa, whereas 15-20 minutes of food restriction appears to reduce
70
the overall protein quantity, exhibiting a protein profile similar to control (un-treated) worms
(Figure 1). In addition, we have seen that exercise and food restriction affect the NL5901 worms
differently downstream — whereas exercise appears to be protective as measured by thrashing
assay scores and BN-Page blot analysis at 24 hours, food restriction appears to result in a
detrimental effect. Here, we have attempted to gain an understanding of the underlying
mechanisms involved.
Previous research has shown that the proteasome is upregulated after recovery from
exercise as well as in response to periods of caloric restriction (Sampaio-Marques et al. 2018;
Tipton et al. 2018). In order to understand if 15-20 minutes of exercise or food restriction may be
activating the proteasome, we treated worms with MG132, an anti-tumor proteasomal inhibitor.
MG132 interacts with the 26S ribosomal subunit and thereby disrupts proteasomal degradation.
Due to the general presence of aggregates in the NL5901 worm, we were careful to overexpose
worms to MG132 as we hypothesized that this may render the state of aggregation irreversible
by exercise and food restriction. Previously, we had determined that a 3-hour treatment with a
different compound (Diphyllin, discussed below) was enough time to allow for an effect to occur
i.e. this time was enough to allow for worms to ingest the compound and for it to have an effect
upon protein species. Using this information for reference, we pre-treated worms with MG132 at
a prior established concentration of 10 uM for 3.5 hours prior to 15-20 minutes of exercise or
food restriction (Martinez et al. 2015) (Figure 15).
71

Figure 15. Pre-treatment of NL5901 worms with MG132 abrogates effects of food restriction,
but not exercise, on 66-<480 kDa range proteins. A. Densitometry analysis of 66-<480 kDa
range proteins shows that MG132 treatment causes a significant increase in 66-<480 kDa range
proteins in food restricted worms (*p≤0.0418), but does not affect protein levels in exercised
worms. B. Representative BN-Page western blot shows that pre-treatment of worms with MG132
10 uM significantly abrogates the reduction of protein quantity in the 66-<480 kDa range in food
restricted worms (green arrow), but does not abrogate the effect of exercise (pink arrow.) (N=3,
n=25.)

Our results show that while MG132 pre-treatment did not abrogate the effect of exercise
upon 66-<480 kDa proteins, it did significantly abrogate the effect of food restriction on these
protein species (Figure 15). Here, food restricted worms treated with MG132 showed the same
levels of 66-<480 kDa proteins as MG132 only-treated worms (densitometry analysis not shown
72
for clarity) and a significantly higher amount of protein as compared to DMSO (vehicle) pre-
treated worms subjected to 15-20 minutes of food restriction. In contrast, exercised worms that
were pre-treated with MG132 were barely discernable from exercised worms treated with
DMSO. This data suggest that the proteosome is activated during food restriction to reduce
protein levels, but does not appear to be activated during exercise under these conditions.
Interestingly, it is also important to note that food restricted worms treated with the
DMSO vehicle show a single band present at ~90 kDa. This band is usually only distinctly
visible in exercised worms (Chapters 1-3). In addition, there is no significant difference of
protein levels between exercised and food restricted worms, which departs from results
previously observed in Figure 1 and other results where worms were not treated with DMSO. We
hypothesize that these results may have occurred due to DMSO treatment on its own. It has be
previously observed that DMSO treatment abrogates paralysis induced by Aβ aggregation in the
GMC101 model of Alzheimer’s disease (Frankowski et al. 2013). This data shows that a
significant improvement in paralysis occurs at 0.25% concentration of DMSO. While our
experiments called for a 0.00833% concentration of DMSO by total volume of NGM plate, this
may still explain the smaller differences we observed between exercised and food restricted
worms previously treated with DMSO and may indicate an interesting relationship specifically to
⍺-synuclein aggregates in the NL5901 worm strain. Indeed, we also observed in a series of other
preliminary experiments with NL5901 worms (data not shown) that even treatment with DMSO
at a similar concentration for 15 minutes or 1 hour reduced protein levels by quite a large margin
as compared to control (un-treated) worms. In reasoning about the protective effects of DMSO in
the GM101 strain, Frankowski et al. showed that gene expression of several protective
mechanisms, such as HSP-70, were upregulated in response to DMSO. Interestingly, published
73
research on NL5901 worms found that HSP-70 did not play a role in directly regulating ⍺-
synuclein inclusion, although an indirect role was proposed (van Ham et al. 2008). Together, this
data may suggest that DMSO may act through a different mechanism and may have a unique
relationship to ⍺-synuclein than previously observed with respect to Aβ. Interestingly, it has also
been shown that DMSO treatment of mouse astrocytes and Artemisia annua shoot cultures leads
to increased ROS production (Mannan et al. 2010; Yuan et al. 2014). Future experiments will
aim to further explore the seemingly protective effect which DMSO treatment appears to have
upon ⍺-synuclein aggregates and whether it invokes mechanisms similar to exercise conditions,
possibly through increased ROS production.
In addition to mechanisms related to the proteasome, we attempted to address the
involvement of ROS. As mentioned previously, ROS is known to be upregulated as a result of
exercise and caloric restriction and levels have been shown to be up-regulated in swimming N2
C. elegans worms (Schulz et al. 2007; Cheng et al. 2016; Laranjeiro et al. 2017; Feng et al.
2020). In addition, ROS is also known to play a role in signaling autophagy in response to
stresses such as glucose restriction (Filomeni et al. 2015).
Previous research has shown that supplementation with anti-oxidants such as N-acetyl-l-
cysteine (NAC) after exercise abrogates induction of signaling mechanisms specifically in the
muscles (Michailidis et al. 2013). Here, we pre-treated worms for 3.5 hours with NAC in order to
observe if treatment may abrogate the effects of exercise upon ⍺-synuclein protein species. We
see that while NAC pre-treatment did not show a statically significant increase in protein levels
(Figure 16A), we noticed that treatment appeared to change the protein profile in the 66-<480
kDa range of proteins (Figures 16 D and E). Unlike previous exercise experiments which have
shown that that there is one predominant species in the 66-<480 kDa region belonging to the
74
monomer (Figures 1 and 2), here we see an increased presence of proteins above the monomeric
band in some experiments, and a general marked difference from the protein profile exhibited in
control H2O (vehicle) exercised worms i.e. the lack of a clear, single band. Additionally, when
quantifying only the region located above the monomeric band, we found that both NAC 5 mM
and 10 mM treated worms showed fold increases in some experiments when this protein region
was compared to exercised (H2O) worms (Figure 16 D and E). Although these ratios were not
consistent across replicates, the changes in the 66-<480 kDa protein profile in the presence of
NAC and exercise appear to demonstrate the involvement of ROS related mechanisms.

Figure 16. NAC pre-treatment shows a significant abrogation of the effects of food restriction on
66-<480 kDa ⍺-synuclein protein species as well as changes in the protein profile of exercised
75
worms. A. Protein quantification of 66-<480 kDa proteins in exercised worms showing a lack of
statistical significance between NAC treated exercised worms and H2O (vehicle) treated
exercised worms for 5 mM and 10 mM concentrations (p=0.1586, p=0.1883, respectively.) B.
Protein quantification of 66-<480 kDa proteins in food restricted worms showing a statistically
significant increase in proteins in NAC 5 mM + FR treated worms (*p≤0.0329) and an almost
significant difference as compared to NAC 10 mM + FR worms (p≤0.0501.) C. Representative
BN-Page gel shows increased presence of 66-<480 kDa proteins in NAC + FR treated worms
(indicated with green arrow) as well as increased protein in exercised worms (indicated by pink
brackets.) D-E. Ratios of proteins in NAC 5 mM pre-treated worms (D) and NAC 10 mM pre-
treated worms (E) as compared to respective H2O (vehicle) control exercised worms. Images of
blots from each replicate next to ratios indicate that NAC effects changes in the 66-<480 kDa
range protein profile in exercised worms. (N=3, n=~25 worms per replicate.)

Interestingly, our results show that food restricted worms pre-treated with NAC at 5 mM
show a statistically significant increase in protein quantity when compared to control (H2O) food
restricted worms, thus implicating ROS in both exercise and food restriction (Figure 16 B).
There was also an almost significant increase with respect to NAC at 10 mM pre-treated food
restricted worms (p=0.0501.) At this time, we did not observe significant effects upon 720 kDa
and well proteins.
Altogether, our results show that NAC pre-treatment changes the protein profile of the
66-<480 kDa region in NAC-treated exercised worms, as well as abrogates the effects of food
restriction. While we are not able to observe the same degree of an effect in NAC pre-treated Ex
worms as we observed in NAC pre-treated FR worms, the deviation from the usual presence of a
single band at ~90 kDa, which we have observed in multiple previous biological replicates
(Figure 1), indicates abrogation of Ex mechanisms. From these results, we hypothesize that the
downstream protective effect of exercise in relationship to ⍺-synuclein aggregation may stem
from the interaction of the predominantly monomeric species present after exercise with ROS.
This leads to the formation of ⍺-synuclein species which resist aggregation over time, leading to
results observed in Chapter 3; Figure 14, where exercised worms contain less higher molecular
76
weight protein species than food restricted worms. Conversely, interaction of ROS with various
different oligomeric species present after food restriction, leads to the formation of species that
are pro-aggregative as well as insoluble/non-degradable under the protective conditions of
exercise (Schematic 2). Aside from direct chemical interactions, our results in Figure 16 may
also suggest that ROS is responsible for signaling autophagic mechanisms and subsequent
protein degradation. In order to further understand the effect which ROS may have on ⍺-
synuclein species, future experiments will aim to utilize mass spectrometry to observe presence
of methionine oxidation or di-tyrosine bonds in ⍺-synuclein species belonging to Ex or FR
worms.

Schematic 2: Representation of current hypothesis founded upon divergent downstream effects
of exercise and food restriction in NL5901 worms.

77
II. Exploring the therapeutic effect of a novel HIF-1⍺ stabilizing compound, Diphyllin
(DP.)
During exercise, hypoxia is increased in the GI tract because of blood re-allocation to
other organs such as the heart, lungs, and muscles. This change induces the hypoxia inducible
factor-1alpha (HIF-1⍺). In response to low oxygen stress, HIF-1⍺ is stabilized in the cytosol and
dimerizes with HIF-1β, a constituently expressed protein. The dimer translocates to the nucleus
where it binds to hypoxia response elements (HREs), upregulating a variety of different
mechanisms. Among these downstream targets are a variety of neuroprotective genes such as
heme oxygenase-1 (HO-1), erythropoietin (EPO), and vascular endothelial growth factor
(VEGF), as well as BCL2 Interacting Protein 3 (BNIP3), a regulator of autophagy and
mitophagy. Interestingly, recent research has shown that HIF-1⍺ is induced in the
gastrointestinal system during exercise (Wu et al. 2020). As it has been hypothesized that
Parkinson’s may begin decades before motor symptoms appear via ⍺-synuclein aggregation in
gut epithelial tissues, induction of HIF-1⍺ and downstream mechanisms of autophagy, may be
particularly relevant as a corrective therapeutic approach to Parkinson’s.
In order to explore HIF-1⍺’s neuroprotective role, we utilized a cell reporter assay
containing a luciferase tagged HIF-1⍺ construct and identified Diphyllin (DP) as a strong inducer
of HIF-1⍺ protein levels. Here, we provide a summary of our results on the effects of DP in
neuroprotective assays in differentiated SHSY5Y cells as well as the NL5901 and GMC101 C.
elegans worm models of Parkinson’s and Alzheimer’s proteotoxicity, respectively.

i. Natural Product Library (NPL) 640 screen for the identification of HIF-1⍺ stabilizing
compounds.

78
In a study analyzing post mortem Parkinson’s brain tissues, researchers found that PD
tissues exhibited a lower level of HIF-1⍺ protein and an increased presence of Prolyl
Hydroxylase Domain Protein 2 (PHD2) (Grnblatt et al. 2004; Elstner et al. 2011; Chiang et al.
2020). Furthermore, previously published work from the Andersen laboratory has showed that
HIF-1⍺ induction of ATP13A2, a p-type ATPase found to be mutated in a young onset form of
Parkinson’s called Kufer-Rakeb syndrome, constitutes a promising novel target for the
prevention or slowing of neurodegeneration associated with Parkinson’s disease (Rajagopalan et
al. 2016). In order to harness HIF-1⍺ therapeutic potential, the Andersen laboratory initiated a
search for natural product compounds using an in vivo human dopaminergic cell reporter line
containing a HIF-1⍺ luciferase construct developed by collaborators (Smirnova et al. 2010).
Here, 85,000 synthetic compounds were screened in a high-throughput manner and chemical
modeling software was used to detect chemical moieties responsible for interacting with the
PHD2 ligand binding site. Compounds were found to inhibit PHD2 via specific interactions with
the iron (Fe
2+
) center and surrounding amino acids.
The reporter system consists of the human dopaminergic SHSY5Y cell line expressing a
HIF-1α Oxygen Degradable Domain (ODD) — amino acids 530-653 — tagged to luciferase
(Smirnova et al. 2010). The ODD contains prolyl 564, one of two HIF-1⍺ prolyl residues that are
hydroxylated by PHD2 under basal conditions leading to HIF-1α degradation. The second prolyl
at position 402, which is hydroxylated following 564 under higher oxygen conditions, was not
included in this construct. Using this in vivo reporting system, we performed a high-throughput
screen (HTS) of Natural Product Library (NPL) 640 and identified Justicidine B as our top hit
(Figure 17A). However, due to lack of availability, we tested a close homolog, Diphyllin (DP),
which contains an additional hydroxyl group (Figure 17B) and found that DP significantly
79
induces HIF-1⍺ luciferase levels (Figure 17C). Here, we see that DP induces HIF-1⍺-luciferase
levels at a range of concentrations, from 0.5 uM to 50 uM, increasing levels significantly
compared to control (DMSO) treatment and a positive control, ciclopirox (CP) utilized in
previous research (Smirnova et al. 2010). The highest levels of induction occur around
concentrations 1.5, 2.5 uM and DP 5 uM (highlighted by patterned marking), with fold increases
ranging from about 4 to 5-fold as compared to control (DMSO).
In addition to these results, we analyzed all of our top natural product hits and compared
their chemical motifs to data previously published from Smirnova et al. Interestingly, Justicidine
B and Diphyllin, differed greatly from previously published results of potent PHD2 inhibitors.
Briefly, Smirnova et al. found that a top hit contains two conjugated benzene rings, one
containing a nitrogen instead of a carbon (quinoline), and the other a hydroxyl group three bonds
away. As electronegative moieties, the nitrogen and hydroxyl groups would interact well with
the Fe
2+
, forming a five membered non-covalent ring with iron. As can be seen in Figure 17A
and B, both Justicidine B and Diphyllin lack this motif. However, we hypothesized that these
compounds could potentially still interact with the ligand binding site in PHD2 perhaps through
interactions between the lactone ring with the iron center (red and purple circles), and the
methoxy functional groups (red and purple arrows) with amino acids surrounding the binding
pocket, specifically the arginine residue at position 322, which was observed to also be important
for one of the highest hits in Smirnova et al.
80

81
Figure 17. Identification of Diphyllin (DP) as a HIF-1⍺ protein stabilizing natural product and
downstream protective effects. A. Chemical structure representation of Justicine B as the top hit
derived from the original NPL 640 HTS screen. The red oval indicates the lactone ring, which
was proposed to interact with Fe
2+
; the red arrow indicates the two methoxy groups. B. Chemical
representation of Diphyllin (DP), a homolog of Justicine B, showing the additional hydroxyl
group (purple arrow) along with the lactone and methoxy groups as shown in B. C. Diphyllin
(DP) induces HIF-1⍺ as observed in the SHSY5Y HIF-1⍺ luciferase cell reporter cell line. DP
concentrations from 0.5 to 50 uM were tested yielding significant induction as compared to
control (DMSO) (indicated by * values) (****p<0.0001) and positive control, Ciclopirox (CP)
(indicated by
#
values) conditions. DP 1.5, 2.5, and 5 uM show the greatest fold-induction of
HIF-1⍺ (
####
p<0.0001; DP 1.0 uM vs CP
##
p≤0.0036; DP 20 uM vs CP
###
p≤0.0002; DP 30 uM
vs CP
#
p≤0.0193; DP 40 uM vs CP
#
p≤0.0321.) D-F. Preliminary results showing treatment of
differentiated human SHSY5Y cells with DP 1.5 uM and CP in the presence of a Paraquat (PQ)
challenge as measured by CyQuant 24 hours post treatment. Each dot represents a technical
replicate. D. Pre-treatment with DP 1.5 uM significantly protects SHSY5Y cells against PQ
(****p<0.0001.) DP 1.5 uM also shows a significantly higher cell viability as compared to
control (DMSO) (*p≤0.0111); PQ 500 uM significantly reduces cell viability as compared to
control (DMSO) (*p≤0.0299); DP 1.5 uM with PQ challenge shows significantly higher values
(***p≤0.0001.) E. Pre-treatment with CP 5 uM significantly protects cells from PQ but to a
smaller extent that DP 1.5 uM (*p≤0.0187.) CP also significantly induces cell viability
(**p≤0.0023.) Unlike in DP 1.5 uM treated cells, there is no significant difference between CP 5
uM in the presence of PQ (p=0.2052.) F. Pre-treatment with CP 10 uM significantly protects
cells from PQ but to a smaller extent than DP 1.5 uM (*p≤0.034.) Unlike in DP and CP 5 uM
treated cells, there are no significant differences between CP 10 uM and control (DMSO) and
between CP 10 uM in the presence of PQ and control (DMSO) (p=0.2598 and p=0.2806,
respectively.) (N=2, n=8-10.) G-I. Two hours pre-treatment with DP 1.5 uM and CP 10 uM
shows significant protection against PQ in SHSY5Y 48 hours post-treatment in human SHSY5Y
dopaminergic cells. Each dot represents a technical replicate. G. Pre-treatment with DP 1.5 uM
shows a significant protection against PQ toxicity (***p≤0.0001.) PQ 500 uM at 48 hours shows
a significant reduction of cell viability to ~60% as compared to control (DMSO) (***p≤0.0002.)
DP 1.5 uM shows an almost significant increase in cell viability as compared to control (DMSO)
(p=0.052.) DP 1.5 uM in the presence of PQ shows no statistical difference from control
(DMSO) (p=0.2516.) H. Pre-treatment with CP 5 uM shows an almost significant protection
against PQ toxicity (p=0.0844) despite showing a statistically significant increase in cell viability
on its own (***p≤0.0006.) CP 5uM in the presence of PQ shows an almost statistically
significant decrease is cell viability as compared to control (DMSO) (p=0.0767.) I. Pre-treatment
with CP 10 uM shows significant protection against PQ toxicity (****p<0.0001.) CP 10 uM
shows a significant increase in cell viability (***p≤0.0009.) CP 10 uM in the presence of PQ
also shows a statistically significant increase as compared to control (DMSO) (*p≤0.0.0222.)
(N=2, n=7-12.) J-L. Preliminary results showing qPCR analysis in differentiated SHSY5Y cells
in response to DP 2.5 uM treatment. Each dot represents a technical replicate. J. Induction of
HO-1 levels (a surrogate for HIF-1⍺ protein levels) is observed between 1 to 2.5 hours post-
treatment as compared to control (DMSO) cells. A significant reduction in gene transcription
levels is seen at 24 hours post-treatment as compared to 1, 1.5, 2, and 2.5 hours pos-treatment
(*p≤0.0129, **p≤0.0016, ***p≤0.0.0006, *p≤0.0493 respectively.) K. HIF-1⍺ gene transcription
levels show values close to control (DMSO) between 1 and 2.5-hours post-treatment, and show a
82
comparatively significant increase at 24 hours post-treatment to levels slightly above control
(DMSO) (**p≤0.0074, *p≤0.0049, and **p≤0.0032 respectively.) L. BNIP3 gene transcription
values are near control (DMSO) levels between 1 and 2.5 hours post DP treatment but are
significantly increased between 2 and 3-fold 24 hours post-treatment (***p≤0.0005,
***p≤0.0003, ***p≤0.0004, and ***p≤0.0003 respectively.) (N=2, n=3.)

In order to better understand the possibility of this interaction, we initiated collaborations
with Dr. Christopher Schofield (Cambridge UK), a chemist with expertise in the PHD2-HIF-1⍺
signaling pathway, and Dr. Dimitry Hushpulian, a co-author in the Smirnova et al. paper.
Interestingly, neither lab was able to find evidence for a direct interaction of Justicidine B nor
DP with the ligand binding site in PHD2. However, both noted that this does not mean that HIF-
1⍺ proteins are not stabilized. Specifically, it is possible that these compounds either interact
with a different protein along the HIF-1⍺ degradation pathway, such as VHL, or, perhaps interact
with a different area in PHD2 outside of the binding pocket, such as one of PHD2’s “legs.”
After observing its ability to stabilize HIF-1⍺ protein levels, we aimed to observe DP’s
neuroprotective capability. Here, we treated differentiated SHSY5Y cells with DP 1.5 uM and
paraquat (PQ) 500 uM. This concentration of DP was chosen due to concerns over cell toxicity
(a further discussion of some obstacles encountered in cell viability assays is discussed below).
We observed cell viability with the CyQuant assay, a two-component dye solution where one of
the dyes is permeable to live cells, passing through the nuclear membrane and interacting with
DNA, thereby causing the cells to fluoresce, while the second dye, is only permeable to dead
cells and therefore acts as a background. Here, our preliminary results show that at 24 hours
post-DP treatment, DP 1.5 uM exhibits slightly greater cell viability values in compound alone
conditions, and a significant protection against PQ 500 uM (Figure 17D). These results indicate
that DP may be causing cell proliferation in our cell culture model. In comparison, the positive
control, CP 5 mM, shows a slightly greater degree of cell proliferation, but a slightly smaller
83
degree of protection against PQ, although still statistically significant (Figure 17E). In
comparison, CP 10 uM does not appear to induce cell proliferation at 24 hours, yet retains a
similar degree of protection against PQ (Figure 17F).
It is important to note that PQ 500 uM does not reach the degree of cell death usually
observed (around 40-50%). In order to observe if DP can exhibit protection against a stronger PQ
challenge, we also assayed the same cells at 48 hours post-treatment (Figure 17 G-I). Here, cell
viability was significantly decreased, to approximately 60%. In these results, we observed that
while cell viability in compound-alone treatment did not quite reach significance (p=0.0520), DP
was still able to strongly protect cells against PQ treatment, maintaining values very close to
control (DMSO) (Figure 17G). Interestingly, we observed a difference in the effects of CP 5 and
10 uM than previously observed at 24 hours — while CP 5 mM showed a significant increase in
cell viability as compared to control (DMSO), it only showed a trend towards a significant
protection in the presence of PQ (p=0.0844) (Figure 17H). On the other hand, CP 10 uM showed
a significant increase in cell viability as compared to control (DMSO), and a significantly greater
protection in the presence of PQ, where cell viability was also significantly greater than values in
control (DMSO) (Figure 17I). These results are converse to our observations in Figures 17 D and
F, where CP 5 uM appeared to be a better concentration at 24 hours than CP 10 uM.
In addition to the luciferase cell reporter system which determined that DP can stabilize
HIF-1⍺ protein levels, we investigated further if DP can induce gene transcription of heme-
oxygenase 1 (HO-1), a downstream target of HIF-1⍺, which has been shown to be
neuroprotective. Because HIF-1⍺ is tightly regulated, HIF-1⍺ protein levels are difficult to assess
directly and HO-1 is often utilized as a surrogate. Here, our preliminary results show that DP 2.5
uM treated differentiated SHSY5Y cells show an increase in HO-1 levels at 30-minute intervals
84
between 1 and 2.5 hours to approximately 3-fold as compared to control (DMSO) (not shown)
(Figure 17J). However, at 24 hours, HO-1 levels decrease to below 2-fold as compared to levels
at times from 1 to 2.5 hours.
In addition to HO-1 levels, we also assessed the effect of DP on the HIF-1⍺ gene (Figure
17K). Although DP should work by stabilizing protein levels as determined in the cell reporter
assay described above, we also checked if any changes may be occurring at the gene level in
order to determine if any positive or negative feedback may be present. Here, we observed that
between 1 and 2.5 hours, HIF-1⍺ levels were below control levels, while at 24 hours, HIF-1⍺
levels significantly increased when compared to 1.5 and 2.5 hours, and almost significantly when
compared to values at 1-hour post-treatment (p=0.0543). These results indicate that a negative
feedback loop may be present in the earlier treatment times but that levels appear to be stable at
24 hours. Furthermore, it appears that HIF-1⍺ gene levels seemed to alternate every 30 minutes,
although these changes were not significant.
In addition to these genes, we also observed the effect of DP treatment on another
previously mentioned HIF-1⍺ target, BNIP3, a regulator of mitophagy and autophagy (Lu et al.
2018). Here, our results show that while BNIP3 levels remained near control levels between 1
and 2.5-hours post-treatment, there was a significant induction in gene expression to 2 to 3-fold
induction 24 hours post-treatment (Figure 17L). This data suggests that longer treatments are
required for autophagy induction in differentiated SHSY5Y cells.

ii. Experimental obstacles encountered in neuroprotective assays with Diphyllin (DP) in cell
culture models of Parkinson’s disease.

85
In our work with DP, we encountered a few interesting challenges which in our opinion,
helped to inform us better of the intricacies involved in working with HIF-1⍺. As HIF-1⍺
induction has been proposed to play a neuroprotective role the context of PD, our goal in this
project was to observe if DP may protect various dopaminergic cell lines against a series of PD-
related neurotoxin models. We primarily worked with 1-Methyl-4-phenylpyridinium ion (MPP
+
),
6-hydroxydopamine (6-OHDA), and paraquat (PQ). However, here we encountered several
challenges in obtaining consistent results among the different assays we tried and will provide a
brief discussion of how we attempted to address these issues.
For our first experiments with DP, we began our work with the N27 rat dopaminergic cell
line and applied the MTT assay as a method for quantifying cell viability in the presence of
MPP+. The MTT assay is a colorimetric assay which measures the turnover of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, a purple insoluble
crystal-like compound, by the mitochondria. This assay has been commonly used as a method for
quantifying cell viability, a readout proportional to mitochondrial health.
In our experiments, we utilized three different DP concentrations — 0.5, 1.5, and 2.5 uM,
and cells were pre-treated with these concentrations for approximately 2 hours before the
addition of the MPP+ neurotoxin. MPP
+
is the ionic form derived from 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP). When MPTP crosses the blood-brain-barrier (BBB), it is
converted to the active reagent, MPP
+
, by monoamine oxidase (Langston 2017). Interestingly,
MPP
+
was discovered in the late 1970’s when a mysterious case of Parkinsonism affected
individuals who ingested a batch of street made heroin. It was reported that the symptoms of
these individuals appeared overnight, and led to the discovery of MPP
+
as a very strong inducer
of Parkinson’s neuropathology in cell and animal models (Langston 2017). It has been reported
86
that MPP
+
likely causes cell death by disrupting Complex I and thereby decreasing cell
respiratory capacity and depleting ATP production. It is thought to specifically attack
dopaminergic cells because it is well recognized by the dopamine receptor. In our assays, we
utilized MPP+ to induce cell toxicity.
In the MTT assay, lower MTT values i.e. lower turner over of MTT to formazan, indicate
lower mitochondrial numbers and therefore poor cell health, possibly even death. We
hypothesized that induction of HIF-1⍺ by DP would protect N27 cells, yielding higher MTT
values. Interestingly, we observed that in cells with drug alone, or with drug and toxin, DP
sometimes show significantly reduced MTT values. While these results would normally indicate
that DP reduces cell viability, we noticed a discrepancy when we observed cells under the
microscope — DP treated cells appeared to look healthier (more transparent and fuller) than cells
with toxin alone. In reference to our previous results observing that BNIP3 upregulation occurs
24 hours post DP treatment, these results suggest that any readings (which were also performed
24 hours post-treatment) showing decreased MTT values, may result from the induction of
mitophagy.
As the morphology of the cells indicated that DP treatment may indeed be beneficial in a
toxic environment despite the low MTT values, we continued to explore DP’s neuroprotective
potential, this time with a different neurotoxin, 6-hydroxydopamine (6-OHDA), which also acts
on dopaminergic cells, where it can inhibit mitochondrial Complex I as well as autoxidize and
form ROS (Simola et al. 2007). Unlike in our previous results with MPP
+
, DP did not exhibit
lower MTT values with or without toxin, but interestingly showed an increase in values at 2.5
uM. However, given our previous observations with MPP+, we cannot ascertain if DP induces
87
cell proliferation. Counter to this result suggesting cell proliferation in DP 2.5 uM treated cells,
DP was not able to protect cell against 6-OHDA toxicity.
Upon experiencing continued variability in this manner, we attempted to troubleshoot by
utilizing different DP concentrations such 1, 3.75, 5, and 10 uM. In addition, as pre-treatment
time can also be considered a form of drug-dosage, we attempted to expose cells to both shorter
as well as longer treatment periods, such as 1,1.5 hours versus 3, 4 hours. However, we were still
unable to obtain any protective effects despite these changes.
Lastly, we also considered that as a natural product chemical, DP may be less stable and
have a shorter half like than perhaps synthetic compounds. We addressed this possibility by
testing old and new batches of DP in the HIF-1⍺-luciferase reporter cell line, but ultimately, saw
very similar levels of HIF-1⍺ induction between these sources. This likely indicates that the
stability of the chemical is not a strong contributing factor to the variable results we observed in
our experiments.
In order to address to understand the best way in which to work with DP, we began to
consider several driving factors that may be causing these results. We hypothesized that one
source may stem mainly from the nature of the HIF-1⍺ pathway. The HIF-1⍺ pathway regulates
a very important physiological response to low oxygen concentrations, allowing the body to
survive these harsh and stressful conditions. For this reason, HIF-1⍺ protein levels are highly
regulated. We hypothesize that when altering this pathway by inducing HIF-1⍺, we may have 1)
upregulated compensatory mechanisms, 2) the environment was not conducive to sustain high
levels of HIF-1⍺ i.e. presence of molecular oxygen under normoxic conditions allowed PHD2 to
continue degrading HIF-1⍺ protein levels, which may especially be possible if DP does not
88
directly inhibit PHD2 as our collaborators have suggested, and/or 3) that continuous HIF-1⍺ was
detrimental to our model systems.
With this in mind, we believe that dosage is likely a very important factor with regard to
the HIF-1⍺ pathway and we may have experienced issues when chronically driving this
mechanism as discussed further in Section III. Indeed, we hypothesize that inducing the HIF-1⍺
pathway can be a very strong and effective therapy against Parkinson’s but that it is probably
most effective when provided in small doses i.e. should be considered a hermetic treatment. In
future experiments, we hope to explore this relationship further. Specifically, we will aim to test
the effect of shorter dosage times, either through pharmacological means with DP or known HIF-
1⍺ inducers, or perhaps in the form of hypoxia therapy, which can be achieved by exposing cells
or worms to low oxygen conditions in specialized chambers. Excitingly, a current funded study
from the Michael J. Fox foundation is studying the effect of hypoxia treatment in PwP.
In addition, we considered that another source of variability may stem from our choice of
cell lines. Specifically, we wondered if perhaps the immortalization of the cell line through
cancerous pathways was an important contributing factor to our results. Although the cell lines
we have used effectively model dopaminergic neurons and have led to discoveries of novel
treatments and mechanisms related to Parkinson’s disease, they may not be ideal for observing
mechanisms related to HIF-1⍺ specifically, because of the effects of hypoxia on cancer. It has
been shown that the most dangerous forms of cancer contain tumorous cells with elevated HIF-
1⍺ protein levels (Soni and Padwad 2017). This may explain why in some experiments we
observed DP inducing cell proliferation. Conversely, DP is utilized specifically as an anti-cancer
agent (Paha et al. 2019; Chen et al. 2018 Jan 4). This latter result may explain why we also
89
observed a decrease in cell viability at times. While it is still difficult to understand our results
overall, the relationship between HIF-1⍺ and cancer is an important point to consider.
In addition to all forementioned hypotheses, we have also considered that another source
of variability may come from DP’s possible mechanism of action — DP has been found to be a
potent inhibitor of v-ATPases, promoting HIF-1⍺ protein stabilization through reducing
concentrations of Fe
2+
in the cytosol. This decreases PHD2’s ability to hydroxylate HIF-1⍺ and
helps to stabilize protein levels (Sørensen et al. 2007; Miles et al. 2017; Duan et al. 2020).
According to this, DP would not directly inhibit PHD2 and as a result, there would be several
mechanisms in between DP’s point of action and PHD2 inhibition, which may introduce sources
of variability among the different actors in this mechanism.

III. DP treatment of C. elegans models of Parkinson’s and Alzheimer’s proteotoxicity.

In order to test DP’s neuroprotective effects in an in vivo PD proteotoxicity model,
NL5901 C. elegans worms were pre-treated with DP 4 uM for 3 hours prior to exercise and food
restriction as described above. Tissue samples were analyzed using BN-Page gels. In addition,
thrashing assays were performed on Day 1 worms pre-treated with DP for 3 hours, and
subsequently subjected to 3 hours of swimming exercise. Thrashing abilities were also measured
on Days 3 and 6 of adulthood. Additionally, GMC101 worms were also treated with DP and the
effect on paralysis scored.

i. Effect of DP 4 uM treatment on native ⍺-synuclein protein species in NL5901 worms.
Prior to beginning treatments in C. elegans, we determined the safety of DP for worms by
growing worms on DP 2.5, 5, and 10 uM treated plates. From this assay, we found that DP 2.5
90
and 5 uM did not cause any developmental delays or lethality, while DP 10 uM caused both side-
effects. After a few thrashing assay experiments with DP 5 uM, we found that this concentration
seemed to exhibit some toxicity as well, and instead found that DP 4 uM was more optimal. In
order to observe the effect of DP treatment on native human ⍺-synuclein protein species,
NL5901 worms were treated with DP 4 uM for 3 hours.
Here, preliminary results show that a 3-hour treatment time significantly reduces well
proteins when compared to control (DMSO) worms (Figure 18). We did not observe significant
differences between DP treated and control (DMSO) worms in the 66-<480 kDa and 720 kDa
proteins (data not shown). In addition to the 3-hour DP treatment, worms were subjected to
exercise and food restriction conditions for 15-20 minutes and were compared with worms that
were also pre-treated with DMSO (vehicle). Here, we see that there is a significant difference
between DP alone and DP + FR worms. In addition, while not statistically significant, our data
indicates that differences between DMSO + FR and DP + FR is rather large in well proteins,
which may indicate that FR works together with DP to further reduce these proteins (p=0.1154).
Future experiments will aim to determine if DP enhances the effects of FR and improves
thrashing outcomes, as well as reducing the possibly detrimental effects upon ⍺-synuclein
protein species 24 hours post-treatment as previously observed in Figures 10, 11 and 14.
91

Figure 18. Preliminary results showing that 3 hours treatment with Diphyllin (DP) in NL5901
worms significantly reduces the quantity of well proteins. A. Well protein quantification of
control (DMSO), DMSO + Ex, DMSO + FR, DP (4 uM), DP 4 uM + Ex, and DP 4 uM + FR
worms. DP 4 uM treated worms show a significantly reduced quantity of well proteins
(*p≤0.0426.) Worms pre-treated with DP 4 uM and subjected to food restriction conditions (DP
4 uM + FR) also showed a significant decrease in proteins as compared to DP 4 uM
(**p≤0.0088.) B. Representative BN-Page blot showing reduction of well proteins upon DP and
DP + FR treatments (purple and green arrows, respectively.) (N=2, n=25 worms per condition.)

ii. Effect of DP 4 uM treatment on NL5901 thrashing assays.
In our initial experiments with DP treated NL5901 worms, which were performed prior to
exploring the effect of swimming exercise upon these animals, we observed worms mainly on
Days 5 and 8, following a consecutive treatment with DP beginning on Day 1. However, we
began to observe that Day 5 worms treated with DP appeared to look less healthy than control
(DMSO) worms — worms would appear to be quite fragile and a subset would die. We noticed
92
that these worms were experiencing egg laying defects — accumulation of eggs — which was
likely the source of the problem. Although we were initially hesitant to treat worms with FUdR
to alleviate this issue because of unknown interactions which could occur with DP, we became
aware that constant upregulation of HIF-1 results in the egg lay defect, and as a result, began
utilizing FUdR (Pender and Horvitz).
As mentioned before in Chapter 3, we also observed that the common method of
performing thrashing assays — picking worms into a drop of liquid, allowing worms to
equilibrate for 30 seconds, and then counting thrashing patterns for the consecutive 30 seconds
using a hand clicker — was perhaps not ideal for the NL5901 strain as we noticed a relatively
large amount of variability between worms. In order to attempt to reduce variability, we followed
advice suggesting the introduction of an increased period of equilibration time in liquid solution.
In fact, these experiments served as a prelude to our exercise studies described above in Chapters
1-3.
After the realization that NL5901 worms required longer periods of equilibration to liquid
solution and that FUdR was required, if we wanted to observe the effect of DP at greater ages we
needed to adapt a different experimental approach than described in the previous Chapters —
Day 1 worms were pre-treated with DP for 3 hours before placement into S-basal for 3 hours.
After this period of time, worms were treated similarly as described previously — they were
placed under a recording microscope and recorded for 30 second intervals. Thrashing counts
were scored later from these video recordings. Although we were not yet aware of the strong
effect which swimming exercise has on ⍺-synuclein proteins at the time these experiments were
performed, we found that DP 4 uM treated worms had a significantly higher thrashing rate than
93
control (DMSO) worms, indicating an ability to further protect worms from ⍺-synuclein
aggregation in addition to the ameliorative effects of exercise described above (Figure 19A).
After experiments were performed on Day 1, all worms were placed back onto DP treated
plates and were observed once again on Days 3 and 6 (Figure 19 B and C). Here, we see that
while DP 4 uM treatment on Day 3 shows a slightly higher thrashing rate than control (DMSO)
worms, this difference is not significant (p=0.4844.) However, we again see a strong and
significantly higher thrashing rate (approximately 4-fold) in DP 4 uM treated worms as
compared to control (DMSO) on Day 6. Here also, DP 4 uM treated worms maintain
approximately the same thrashing rate as seen in control (DMSO) worms on Day 1, and the rates
observed in both groups on Day 3. This suggests that DP treated worms are able to maintain or
reverse the effects of age on the NL5901 worms with regard to ⍺-synuclein aggregation.
However, it is also important to note the lower worm numbers present on Days 3 and 6 as
compared to Day 1. These observations suggest that even in the presence of FUdR, which was
able to alleviate the bagging effect, constant HIF-1 activation is likely not beneficial to the
worms.

94

Figure 19. Diphyllin (DP) significantly improves the thrashing ability of NL5901 worms. Here,
results show improvement in thrashing on Day 1 (A) (N=3, n=~40-60 per condition)
(****p<0.0001) and Day 6 (C) (N=2, ~15-60 per condition) (****p<0.0001.) On Day 6,
preliminary results show that DP treated worms exhibit thrashing rates seen in Day 1 control
(DMSO) worms. Day 3 preliminary results do not show significant differences between DP 4
uM treated and control (DMSO) worms (p=0.4844) (N=2, ~15-60 per condition.)

iii. Effect of DP 4 uM treatment on GMC101 paralysis assays.
In order to observe if the effect of DP can be generalized to other forms of proteotoxicity,
we utilized GMC101 worms. GMC101 worms overexpress human A-β within muscle cells,
resulting in temperature-dependent paralysis which occurs upon transfer of worms from 20°C to
25°C. In our experiments, worms were picked to control (DMSO), DP 2.5 uM, and DP 4 uM
treated plates at the L4 stage and then transferred from 20°C to 25°C. Worms were scored at 24
and 48 hours post transfer by gently picking and placing worms on the edge, or near the edge, of
the bacteria lawn. Worms were lined up carefully and observed after approximately 20 minutes.
95
Worms that remained in their place and that did not readily respond to a gentle touch to their
head by the pick, were scored as paralyzed. In our results, we see that DP 4 uM was able to
mediate an almost significant protection against paralysis at 24 hours, and showed a significant
protection against paralysis after 48 hours (Figure 20).

Figure 20. Diphyllin (DP) protects GMC101 worms against paralysis at 48 hours post treatment.
A. DP 4 uM shows an almost significant protective effect against paralysis at 24 hours post
treatment as compared to control (DMSO) worms (p=0.0848.) DP 2.5 uM does not show a
significant protection (p=0.1079.) B. DP 4 uM shows a protective effect against GMC101
paralysis 48 hours post-treatment as compared to control (DMSO) (*p≤0.0390) and an almost
significant protective effect as compared to DP 2.5 (p=0.0817.) DP 2.5 uM does not show a
significant protection against paralysis as compared to control (DMSO) (p=0.1457) (N=4, n=
~40-150 per condition.)

Control (DMSO), 24 hrs.
DP 2.5 uM, 24 hrs.
DP 4 uM, 24 hrs.
0
20
40
60
80
% Worms NOT Paralyzed per Replicate
p=0.0848
Control (DMSO), 48 hrs.
DP 2.5 uM, 48 hrs.
DP 4 uM, 48 hrs.
0
20
40
60
% Worms NOT Paralyzed per Replicate
✱
p=0.0817
96
IV. Summary: Exploring mechanisms involved in exercise and food restriction and
utilizing a novel natural product compound in proteotoxicity models.

Taken together, the above data confirms that exercise and food restriction act through
different mechanisms. In our results, we saw that while pre-treatment with MG132 abrogated the
effects of food restriction on the 66-<480 kDa proteins, it did not seem to reduce the effect of
exercise on these same species. In addition, we observed that pre-treatment with NAC resulted in
changes to the 66-<480 kDa protein profile in exercised worms, as well as caused a significant
increase in proteins in food restricted worms. These data suggest a role for ROS under both
exercise and food restriction, possibly resulting in induced autophagy and thereby the reduction
in protein quantity. Future experiments will explore the possible role which ROS induction may
play in interacting with ⍺-synuclein protein species and forming protective or pro-aggregative
oxidized ⍺-synuclein protein species as outlined in Schematic 2.
We also performed experiments with a novel HIF-1⍺ natural product compound, DP,
observing that it is able to significantly stabilize HIF-1⍺ protein levels. In our preliminary
results, we found that DP induces HO-1 and BNIP3 gene expression, as well as protects
differentiated SHSY5Y cell against paraquat toxicity. Furthermore, preliminary results showed
that DP treatment of NL5901 worms resulted in a significant reduction in well proteins and,
although not statistically significant, seemed to further reduce well proteins in DP worms also
treated with 15-20 minutes of food restriction, suggesting a synergistic effect, something which
we plan to explore in future experiments. Thrashing assays also confirmed the protective effect
of DP in the presence of prolonged exercise, both on Day 1, as well as on Day 6, as compared to
control (DMSO) worms. In addition, we observed a protective effect in DP treated GMC101
worms, where paralysis was significantly reduced in DP 4 uM treated worms after 48 hours as
97
compared to control (DMSO) worms. Altogether, this data suggests that DP treatment is likely
protective via inducing HIF-1⍺ protein levels and consequentially, autophagy. Future
experiments will explore DP’s mechanistic role further, as well as hypoxic treatments of NL5901
worms.

98
Chapter 5: Future directions.

Schematic 3. Summary of differential effects of exercise and food restriction upon NL5901
worms.

Our research so far has demonstrated the dramatic effect of 15-20 minutes of swimming
exercise upon native human ⍺-synuclein protein species within the NL5901 C. elegans worm
model of Parkinson’s disease. Here, we observed that exercise conditions overall decrease ⍺-
synuclein protein quantity, retaining mainly the monomeric species after 15-20 minutes. In
contrast, while food restriction conditions also significantly decreased most ⍺-synuclein protein
99
species, the species remaining include various oligomers. Here we have hypothesized that
exercise and food restriction ultimately confer different downstream effects due to reactions
between ROS and the ⍺-synuclein species remaining after the respective treatments, yielding
protective oxidized monomers under exercised conditions, versus pro-aggregative oxidized
oligomers under food restriction conditions. In the future, we hope to expand our understanding
of these mechanisms, as well as explore the possibly important roles of the energy molecules,
glucose and lactate, in relationship to ⍺-synuclein aggregation.
In addition to this work, we are also very interested in further exploring the
neuroprotective potential of Diphyllin (DP) as a HIF-1⍺ protein stabilizing compound and
attempting to better understand it mechanism of action. While still elusive, our data suggests that
DP is not involved in PHD2 inhibition as we had initially hypothesized, but we have yet to
explore if DP may work to inhibit the von Hippel Lindau protein. We are also interested in
exploring the possible role of a different, quite intricate, mechanism related to HIF-1⍺ protein
stabilization. Previous research found that HIF-1⍺ induction is possible through the inhibition of
lysosomal v-ATPases (Miles et al. 2017). Here, inhibition of v-ATPases by bafilomycin, a
common autophagy inhibitor, was shown to prevent the transfer of Fe
2+
, one of the co-factors for
PHD2, from the lysosome to the cytosol. The reduction in Fe
2+
levels results in decreased PHD2
activity and subsequently, increased HIF-1⍺ protein stability. In addition, separate research
showed that DP treated bone osteoclasts showed a decrease in bone resorption via inhibition of a
v-ATPase subunit, possibly a3, required for osteoclast attachment (Sørensen et al. 2007). In
addition, other work has demonstrated that DP is able to improve glucose tolerance in obese
mice and suggested that DP may be involved in reducing autophagy (Duan et al. 2020). These
later results suggest a conflicting role in DP’s role in autophagy when contrasted with our
100
findings that have shown an induction of BNIP3, as well as decreased ⍺-synuclein protein
aggregation and improved results in NL5901 and GMC101 behavioral assays. We are therefore
very interested in further exploring the possibly divergent role of DP upon autophagy
mechanisms, as well as DP’s role in glucose metabolism, which has relevance to our studies in
exercise and food restriction.
Finally, we are also very interested in exploring the role of hypoxia and HIF-1⍺ with
respect to exercise and the gastrointestinal system. It has been shown that exercise induces
hypoxia and increases HIF-1⍺ levels in the gut in a mouse model of swimming exercise (Wu et
al. 2020). As a result, while exercise causes increased gut permeability and ROS levels in the
short term, it is believed to confer a protective effect in the longer term, suggesting that it is a
form of hormesis (Mach and Fuster-Botella 2017; Keirns et al. 2020; Ribeiro et al. 2021). Gut
permeability, specifically, appears to occur through ischemia-reperfusion injury. Here, 10
minutes after engaging in exercise, blood flow is dramatically reduced to the gut and reallocated
to other organs, causing ischemia in the GI tract. After exercise cessation, blood flow is rushed
backed to ischemic GI tissues, resulting in injury. Despite these changes, chronic exercise
appears to improve gut permeability by improving the mucus layer, splanchnic (gut) blood flow,
induction of heat shock proteins, and promotion of non-inflammatory microbial growth.
In addition, in C. elegans, swimming exercise alleviated gut permeability in N2 worms
by Days 11 and 15. Furthermore, it was recently observed that an iron storage gene, ftn-1, is up-
regulated in the C. elegans gut in response to neuronal sensing of hypoxic conditions, protecting
worms against Pseudomonas aeruginosa infection (Laranjeiro et al. 2019; Romero-Afrima et al.
2020). Furthermore, it has been observed that ⍺-synuclein is contained within gut
enteroendocrine cells (EECs) and the STC-1 enteroendocrine cell line (Chandra et al. 2017). This
101
brings up the interesting question of how ⍺-synuclein located in the gut may be affected by bouts
of exercise, particularly in response to hypoxia and HIF-1⍺ induction. Interestingly, the ⍺-
synuclein gene contains a hypoxia response element and is one of few proteins continually
transcribed under hypoxic conditions, perhaps suggesting an important connection between ⍺-
synuclein and hypoxia (Koukouraki and Doxakis 2016; Ahmed-Muhsin 2009). As data suggests
that ⍺-synuclein aggregation may contribute to the initial spread of Parkinson’s disease from the
gastrointestinal system, we are very interested in further exploring the possible protective
mechanisms of hypoxia and HIF-1⍺ induction in GI tissues in response to exercise in both C.
elegans and cell culture models.

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Appendices:
Appendix A: Methods
i. Worm Experiments.
NL5901- pkIs2386 [unc-54p::alphasynuclein::YFP + unc-119(+)] worms, containing human
overexpressed ⍺-synuclein protein, were purchased from CGC funded by the NIH Office of
Research Infrastructure Programs (P40 OD010440).: All worms were maintained on nematode
growth medium (NGM) at 20
o
C as previously described (Brenner 1974). Worms were age
synchronized by performing egg lays with ~50 worms per plate for 3 hours. Eggs were grown for
3 days to Day 1 of adulthood. Assays were performed in three separate biological replicates
unless otherwise stated.

Swimming Exercise Experiments:

Three conditions — control (un-treated), exercise, and food restriction — were analyzed per
biological replicate in a single day stacked by three min intervals. Approximately 25 worms were
used per condition. Briefly, Day 1 worms were placed into 1 mL S-basal solution on 10 mm un-
spotted agar plates. Plates were left on the bench top undisturbed (to avoid spilling and un-
intentional stimulus which may alter movement) covered with a foil leaf. After 15-20 mins of
exercise, worms were collected into 1.5 mL Eppendorf tubes and allowed time to sink to the
bottom of the tube (30 seconds to 1 min). Excess liquid was carefully removed, leaving behind
approximately 30 uL of solution. Worms were immediately flash frozen in liquid nitrogen and
then placed onto dry ice before storage at -80°C. For food restricted worms, Day 1 worms were
picked and placed onto a small drop of S-basal solution on top of 10 mm unspotted agar plates,
facilitating transfer and allowing dilution of excess bacteria present during transfer. Worms were
then quickly and carefully spread with a pick onto the plate to prevent swimming. As with
swimming exercise conditions, plates were left on the benchtop undisturbed for the allotted
period of time to match conditions for exercised worms. After 15-20 mins (food restriction
treatment times match exercise treatment times), worms were carefully picked into 30 uL of S-
basal solution in a 1.5 mL Eppendorf tube and immediately flash-frozen in liquid nitrogen to
prevent swimming in the test tube. Samples were stored as described above. For control (un-
treated) worms, Day 1 worms were picked directly from a synchronized population plate,
collected as described for food restricted worms, and stored as described above.

Worms were treated similarly for 1 and 2-hour experiments — all three groups of worms were
left on the laboratory bench top (transfer may have resulted in 1) spilling water solution and 2)
disturbing swimming patterns of worms. In addition, conditions were kept as similar to worms
exercised for 15-20 minutes, and these experiments were run in parallel (i.e. all at the same
time.) All worms were covered with a foil leaf while on the bench top, and were collected and
stored as described above.

Blue-Native Page (BN-Page) Western Blot:

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Flash frozen samples were removed from storage at -80ºC and resuspended in 10 uL of Worm
Lysis Buffer (WLB) containing protease inhibitor cocktail. Samples were thawed to room
temperature and afterwards were placed on ice. Tissues were then lysed using a probe sonicator
(550 Sonic Dismembrator, Fischer Scientific) at 4ºC by slowly increasing the setting dial from 1
to 3 until samples formed a white froth. Samples were then centrifuged at 13,000 rpm (centrifuge
5424, Eppendorf) for 30 seconds. Further sample preparation was performed following
previously published protocols from Goya et al. 2020 and Thermofisher with the specification
that G-250 was added at 25% of the detergent (WLB) concentration. Samples were vortexed
(VWR) for ~9 seconds, spun down ~36 seconds in a hand centrifuge (VWR), and stored on ice
prior to gel loading (Invitrogen.) A 40 uL volume of solution was loaded per well.
After transfer, blots were fixed in 8% acetic acid solution (glacial acetic acid, Sigma) for 15 mins
and then rinsed with DI water. Blots were air dried, washed with methanol and afterwards
blocked for 30 mins in 5% milk-PBST solution. Blots were incubated with 1:500 anti-GFP
antibody (Rabbit polyclonal, Santa Cruz) at 4ºC overnight in a test tube rotator. The next day,
blots were washed and blocked with secondary (goat anti-rabbit, Millipore) for 1 hour at room
temperature and then washed again before visualization using High Sensitivity ECL solution
(Chemiluminescent HRP Substrate, Millipore) on ChemiDoc MP (Biorad). A 60 second time
window with 15 images was used. Protein bands were quantified using ImageJ software and
statistical analysis was performed using un-paired, one-tailed t-test. Approximately 25 worms
were used per condition with a total of eight biological replicates per condition.

SDS-page gels and DSS crosslinking: SDS page gels were run in a similar manner to BN-Page
gels. Briefly, worms were sampled for control (un-treated), exercise, and food restriction
conditions in 30 uL of S-basal, flash frozen in liquid nitrogen, and stored in -80°C. During
experimental workup, samples were thawed to room temperature after the addition of 10 uL of
worm lysis buffer and afterwards sonicated as described above, and spun down in centrifuge as
described above. 5 uL of Laemmli sample buffer was added and samples were vortexed and spun
down using hand centrifuge as described above. 40 uL of sample were added to each well. For
samples with DSS, samples were worked up using the protocol previously described, with the
exception that DSS was added to lysed worm tissues (Bartels et al. 2011).

Confocal Microscopy:

PFA fixation: Worms were subjected to the different experimental conditions (control (un-
treated), Ex, and FR) as described above. After treatment, worms were collected in 1.5 mL
Eppendorf tubes in approximately 30 uL of S-basal solution as above. 1 mL of 2% PFA was
quickly added to the tubes. Worms were incubated in PFA for 30 mins in foil-wrapped test tubes.
Afterwards, test tubes were centrifuged in a hand centrifuge (VWR) until all worms collected at
the bottom. Excess PFA was removed leaving ~30 uL. 1 mL of S-basal was added and worms
were stored at 4ºC.

Glass slide preparation: Worms were pipetted from Eppendorf tubes onto fresh bacterial plates.
Plates were dried in a chemical hood, although over-drying of the worms was avoided. Worms
were picked onto glass slides containing an agar pad (prepared as evenly as possible to avoid
complications with confocal imaging i.e. Z-stack ranges) into a small drop of S-basal solution in
order to facilitate transfer. After all worms were placed on agar, a small drop of Vectashield
114
(Vector Laboratories) was pipetted on top of the worms. Solution containing the worms was
gently swirled with a pick to allow unilateral mixing of Vectashield and to avoid worms
overlapping. Overspreading of worms and removal from Vectashield solution was avoided. A
glass coverslip was quickly affixed with nail polish to avoid drying of worms. Only one
condition was prepared on a slide at a time and slides were prepared only 30 mins to 1 hour prior
to visualization.

Confocal microscopy (Zeiss LSM 780 inverted confocal): Worms were visualized on slides
using 10x and 20x objectives and positions fixed using the Zeiss program. Only the head region
was located. The objective was then changed to 40x oil and appropriate Z-stack extremes were
set. Positions were updated to reflect the new Z-plane and scanning was initiated (approximately
1 hour to 3 hours depending on number or worms and Z-stacks required (depends mainly on
differences in agar pad thickness throughout slide)).

Quantification of puncta: Scans were analyzed in Imaris. Images were processed with
background subtraction values of 1 um. Appropriate thresholding was set to generate surfaces for
puncta. Settings were kept consistent for all conditions and biological replicates. Punta count per
head region, and mean as well as individual puncta surface area values were analyzed. Statistical
analysis was performed using 2-way ANOVA and un-paired, one-tailed t-test.

Thrashing assay:

Approximately 100 Day 1 were selected for each condition (control, exercise, or food restriction)
and worms were handled similarly as described above in swimming exercise experiments. After
treatments, worms were carefully transferred to separately labeled bacterial plates. On Day 2 and
Day 4, 25-36 worms were picked from each bacterial plate and subjected to thrashing assays.
Briefly, worms were placed into 1 mL S-basal solution in unspotted 10 mm agar plates as
described above. As quickly as possible, plates were carefully carried to a microscope camera
(Leica DFC 400) and recorded for 30 seconds at 3 mins and at 20 mins post-immersion in S-
basal solution. Plates were not disturbed for the entirety of the assay. Thrashes were later scored
from recordings. A thrashing movement was identified as when worms exhibited one entire cycle
of head and tail movement. Data was analyzed using mixed effects model and un-paired, one-
tailed t-test.

Re-feeding assay: Worms were subjected to 15-20 minutes of exercise and food restrictions as
described above. Afterwards, worms were placed onto bacterial plates — exercised worms were
transferred by collection in S-basal in Eppendorf tubes, pipetted onto bacterial plates using a
severed pipette, and dried in chemical hood (avoiding over drying); food restricted worms were
gently picked onto bacterial plates. Worms were placed back into 20°C incubators and were
sampled at 1 hour or 24 hours. Control (un-treated) worms were picked directly from egg laying
plates at 1 hour and 24 hours. Data was analyzed using un-paired, one-tailed t-test.

Pharmacological treatments: All compounds — MG132, NAC, and DP — were prepared
according to needed concentrations (10 uM; 5 and 10 mM; and 1.5, 2.5, and 4 uM, respectively)
in autoclaved water. For experiments requiring DMSO controls, solutions were prepared
115
similarly using the appropriate volume matching compound concentrations. Liquid solutions
were added at 130 uL per 10 mm bacterial plate.

Day 1 worms were picked from original egg lay plates and approximately 30-36 worms were
gently transferred to compound plates for 3.5-hour time treatments. Afterwards, 25 worms were
subjected to exercise or food restriction conditions as described above. Samples were afterwards
frozen, and work up performed as described above. Data was analyzed using un-paired, one-
tailed t-test.

GMC101 paralysis assay: L4 worms are placed on drug or control plates as described above and
then transferred from 20 to 25°Celsius. Worms were scored at 24 and 48-hours post-transfer.
Worms were scored by gently picking up and placing on the edge, or near the edge, of the
bacterial lawn. After 20 minutes, worms were observed. Worms that crawled away were not
scored as paralyzed. Worms that did not crawl away and did not readily respond to a gentle touch
to the head were scored as paralyzed. Data was analyzed using un-paired, one-tailed t-test.

ii. Cell culture experiments.

NPL-640 high throughput screen: HIF-1 SHSY5Y luciferase cells were acquired from
collaborators (Smirnova et al. 2010). Cells were plated at a density of 30,000 cells per well in
black bottomed 96-well plates in 100 uL of SHSY5Y cell culture medium (ATCC.) PL640
compounds were added from 10 mM diluted library at a final concentration of 10 uM. Luciferin
was added from Promega luciferase assay kit, and cells were visualized in spectrophotometer
using Luciferase assay settings (Cytation, BioTek.) Data was analyzed using un-paired, one-
tailed t-test.

CyQuant neuroprotective assays: SHSY5Y cells were plated at a density of 8,000 cells per well
in 96-well plates in transparent bottoms and black walls. Cells were differentiated by 10 uM
Retinol treatment for 3 consecutive days followed by 8nM TPP treatment on consecutive 3 days
(total of 6 consecutive days.) On the 5
th
day of treatment, cells were pre-treated with DP 1.5 uM
for 2 hours in medium containing TPP. Afterwards, Paraquat 500 uM challenge was
administered and appropriate DMSP volume was added at this time. Separate plates were
allocated for 24 and 48-hour treatments. At 24 and 48 hours after treatment, CyQuant solution
was prepared and added to plates. Cells were visualized using Fluorescence assay settings
(Cytation, BioTek.) Data was analyzed using un-paired, one-tailed t-test.

qPCR: SHSY5Y cells were plated into 6-well plates at a density of 150,000 cells per plate. Cells
were differentiated as described above, treated with DP 2.5 uM for 1, 1.5, 2, 2.5, and 24 hours
and cell pellets were collected in Eppendorf tubes, removing medium and washing with PBS.
Cells were stored at -80°C. RNA was extracted and cDNA made using kit (Qiagen.) Data was
analyzed using un-paired, one-tailed t-test.

Abstract (if available)

Abstract Full title: Swimming exercise reduces native ⍺-synuclein protein species in a transgenic C. elegans model of Parkinson’s disease and confers downstream protective effects independent of food restriction: exploring exercise as a medicine for Parkinson’s disease. Exercise has been historically recommended to prevent many disease conditions. Intense exercise in particular has been shown to be beneficial for Parkinson’s disease (PD)ㅡstopping and even reversing symptoms in some patients. Recent research in mammalian animal models of Parkinson’s have shown that exercise affects ⍺-synuclein aggregate species, considered to be a hallmark of PD. However, the exact changes in native ⍺-synuclein protein species after exercise and the downstream effects of exercise upon the health of the animals remains unclear. Recently, it was shown that swimming constitutes a form of exercise in C. elegans worms that confers a protective effect in several worm models of tau and Huntington protein neurodegeneration. Here we show that a period of swimming exercise (Ex)ㅡ15–20 minutesㅡdramatically reduces several native human ⍺-synuclein protein species in the NL5901 C. elegans worm model of Parkinson’s. Exercise on Day 1 of adulthood was found to improve motor function measured by the thrashing rate of worms on Day 2 and Day 4 when compared to both control (untreated) and food restricted (FR) worms. Moreover, exercised worms show smaller ⍺-synuclein::YFP puncta on average than food restricted worms as measured by confocal microscopy, as well as significantly less higher molecular weight proteins as measured by BN-Page assays. Here we show that exercise reduces native human ⍺-synuclein levels independent of food restriction in C. elegans NL5901 worms.

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Asset Metadata

Creator Schmidt, Minna Yakov (author)

Core Title Swimming exercise reduces native ⍺-synuclein protein species in a transgenic C. elegans model of Parkinson’s disease and confers downstream protective effects independent of food restriction…

School Leonard Davis School of Gerontology

Degree Doctor of Philosophy

Degree Program Biology of Aging

Degree Conferral Date 2021-12

Publication Date 11/30/2021

Defense Date 08/19/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ⍺-synuclein,C. elegans,Exercise,food restriction,NL5901,OAI-PMH Harvest,Parkinson's disease,protein aggregation

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Andersen, Julie (committee chair), Curran, Sean (committee member), Lithgow, Gordon (committee member), Melov, Simon (committee member)

Creator Email MSchmidt@buckinstitute.org

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC17789616

Unique identifier UC17789616

Legacy Identifier etd-SchmidtMin-10262

Document Type Dissertation

Format application/pdf (imt)

Rights Schmidt, Minna Yakov

Type texts

Source 20211206-wayne-usctheses-batch-901-nissen (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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