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The effects of a fasting mimicking diet (FMD) on mouse models of Alzheimer's and Parkinson's disease
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The effects of a fasting mimicking diet (FMD) on mouse models of Alzheimer's and Parkinson's disease
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THE EFFECTS OF A FASTING MIMICKING DIET (FMD)
ON MOUSE MODELS OF
ALZHEIMER’S AND PARKINSON’S DISEASE
by
FLEUR LOBO
A Dissertation presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY OF AGING)
August 2022
Copyright 2022 FLEUR LOBO
ii
Acknowledgements
I would like to express my sincere gratitude to my PhD Advisor, Dr. Valter Longo for his
mentorship, and support during my PhD degree at USC. I am very grateful for his valuable
advice and encouragement on my research projects as well as during the process of our
manuscript submission. It was my dream to carry out research in the field of neurodegenerative
diseases with a focus on possible treatments and this became a reality under his guidance. It has
been an honor to be part of the Longo Lab.
I would like to thank Dr. Christian Pike, Dr. Julie Andersen and Dr. David Lee for their time,
guidance and thoughtful advice during the PhD committee meetings and for always having my
best interests in mind. Thank you, Dr. Julie Andersen, for meeting with me when I had questions
related to my Parkinson’s disease project. I appreciate you taking the time to help me and steer
me in the right direction. I am also thankful to Dr. Min Wei for his valuable inputs and
discussions about my projects especially when I just began the PhD program.
Thank you Seba, Priya, Jerry, Maura, Mahshid, Amrendra, Jonathan, Giacomo and other lab
members for your camaraderie and insightful discussions on research that made my time at the
Longo lab very memorable.
I was given the opportunity to mentor 9 students during the course of my program and I am
grateful to all of my students for their help with the research. It has been a privilege to mentor
and guide such bright and enthusiastic students.
A special thanks to my good friends Marilena and Alexey for always being there for me. When I
worked late on weekdays and had to go in on the weekends to the vivarium, Alexey made sure I
was home safe by walking me home.
iii
I am extremely grateful for my parents, Gerard and Rosita, brother Robin and grandmother
Marie, for always believing in me, and their constant support especially when the road was
tough. Without their encouragement, I would have found it hard to pursue my dreams, leave the
comfort of our home and work towards a PhD degree in another country. They taught me to be
strong and gave me the freedom to follow my own path.
I am so grateful for meeting my fiancé Prashan a few years ago while being a PhD student. He
has been a great source of support and always encourages me to be my best.
Finally, I will always remember my late uncle Gerard D’Souza who believed that I would
complete a PhD degree even before I started the program and would affirmatively use the prefix
Dr. before my name. Thank you for inspiring me to never give up on my dreams.
Figure Credits
1. Ki67
+
Sox2
+
staining and analysis (Figure 3 A-C), CD11b IHC staining and analysis (Figure 9
A-F), Iba1 staining and analysis results (Figure 13 F and G) from Priya Rangan, PhD. (Former
PhD student, Longo Lab)
2. 3xTg-AD/Nox2-KO and Apocynin mouse models, NOR and Y maze behavioral test, and IHC
(Figure 15A-H) from Edoardo Parrella, PhD. (Former Post-Doc, Longo Lab)
3. All the remaining figures except the ones mentioned above (Fleur Lobo)
iv
Data from chapters 1-2, are included in a manuscript that is provisionally accepted at Cell
Reports, under the title “Fasting-mimicking diet cycles reduce neuroinflammation to attenuate
cognitive decline in Alzheimer’s models,” (Priya Rangan
+
, Fleur Lobo
+
, Edoardo Parrella
+
,
Nicolas Rochette, Marco Morselli, Terri-Leigh Stephen, Anna Laura Cremonini, Luca
Tagliafico, Angelica Persia, Irene Caffa, Fiammetta Monacelli, Patrizio Odetti, Tommaso
Bonfiglio, Alessio Nencioni, Martina Pigliautile, Virginia Boccardi, Patrizia Mecocci, Christian
J. Pike, Pinchas Cohen, Mary Jo LaDu Matteo Pellegrini, Kyle Xia, Katelynn Tran, Brandon
Ann, Dolly Chowdhury, Valter D. Longo. Fasting-mimicking diet cycles reduce
neuroinflammation to attenuate cognitive decline in Alzheimer’s models. Cell Reports. In press.)
+
These authors contributed equally to this work.
Data reported in Figure 1 A-E, Figure 2 A-D, Figure 3 A-C, Figure 4 A-B, Figure 5 A-B, Figure
7 A-E, Figure 9 D-F, Figure 11 D-E, Figure 12 F, Figure 14 A-B, Figure 15 A-C were previously
published in the dissertation, “The effects of a Fasting-mimicking diet (FMD) on gastrointestinal
and neurodegenerative disorders,” by Priya Rangan at USC (Rangan 2019).
v
TABLE OF CONTENTS
Acknowledgements ...................................................................................................................... ii
List of Figures ............................................................................................................................... vii
Abbreviations ............................................................................................................................... ix
Abstract ........................................................................................................................................ xii
Chapter 1: Dietary Interventions in Alzheimer’s Disease (AD) ................................................... 1
1.1 Pathogenesis of Alzheimer’s disease ....................................................................... 1
1.2 Current treatments for Alzheimer’s disease ............................................................. 2
1.3 Animal models of AD ............................................................................................... 2
1.4 Alzheimer’s disease and Dietary restriction ............................................................. 3
1.5 Hypothesis and Research Design .............................................................................. 5
Chapter 2: The effects of a Fasting Mimicking Diet on cognition, pathology, neurogenesis,
neuroinflammation and NADPH Oxidase (Nox2) in E4FAD and 3XTGAD mouse models
of Alzheimer’s disease. ................................................................................................................. 7
2.1 Abstract ................................................................................................................... 7
2.2 Results ..................................................................................................................... 8
2.3 Discussion ............................................................................................................... 37
2.4 Materials and Methods ............................................................................................ 40
Chapter 3: Dietary Interventions in Parkinson’s disease (PD) .................................................... 56
3.1 Pathogenesis of Parkinson’s disease ........................................................................ 56
3.2 Autophagy, Mitophagy and Parkinson’s disease ..................................................... 59
3.3 Current Treatments and disease modifying therapies .............................................. 60
vi
3.4 Animal models of PD ............................................................................................... 61
3.5 Diet and PD models. .................................................................................................. 63
3.6 Hypothesis and Research Design .............................................................................. 69
Chapter 4: The impact of Fasting Mimicking Diet cycles on motor-coordination, Alpha
Synuclein aggregates, neuroinflammation and microglial activation in an Alpha Synuclein
overexpression mouse model of Parkinson’s disease. .................................................................. 71
4.1 Abstract ..................................................................................................................... 71
4.2 Results ....................................................................................................................... 72
4.3 Discussion .................................................................................................................. 89
4.4 Materials and Methods ............................................................................................... 92
Chapter 5: Conclusion .................................................................................................................. 99
5.1 Summary of findings and significance ..................................................................... 99
References .................................................................................................................................. 102
vii
List of Figures
Schematic 1. Molecular mechanisms underlying Parkinson’s disease. ........................................ 58
Schematic 2. Mammalian Autophagy Pathway. ........................................................................... 60
Schematic 3. Percentage of macronutrients in Human Prolon versus the Control diet ................ 72
Schematic 4. Summary of the effects of FMD on AD mouse models. ...................................... 100
Schematic 5. Summary of the potential effects of FMD on the ASO mouse model of PD. ...... 101
Figure 1. FMD cycles improve spatial memory in female E4FAD mice. .................................... 9
Figure 2. FMD cycles reduce hippocampal and cortex Aβ load, in female E4FAD mice. .......... 10
Figure 3. The effects of FMD cycles on neurogenesis and the number of astrocytes in the
dentate gyrus of female E4FAD mice. .......................................................................................... 12
Figure 4. Assessment of CD11b-Aβ42 binding in detergent-soluble/triton-soluble cortex of
female ~7-7.5-month-old E4FAD mice ........................................................................................ 13
Figure 5. FMD cycles reduce Nox2 levels in E4FAD mice. ....................................................... 14
Figure 6. Body weight profiles of 18.5-month-old 3xTg-AD male and female mice after
the first four weeks, at the mid-point and end-point of the long term dietary treatment. ............. 16
Figure 7. FMD cycles slow the progression of AD-associated pathology in aged 3xTg mice. .... 18
Figure 8. FMD cycles slow the progression of AD-associated pathology in E4FAD, aged and
8.5-month-old 3xTg mice. Related to Figure 2 and 7. .................................................................. 19
Figure 9. FMD cycles regulate CD11b-immunoreactive (CD11b-ir) microglia levels and
activation in aged 3xTg mice. ...................................................................................................... 21
Figure 10. FMD regulate CD68 positive microglia in aged 3xTg mice ....................................... 21
Figure 11. FMD cycles do not improve spatial memory in female 3xTg mice assessed at
17.5-18 months of age. .................................................................................................................. 23
Figure 12. FMD cycles do not improve spatial memory in male 3xTg mice assessed at
17.5-18 months of age. ................................................................................................................. 24
viii
Figure 13. Short- term treatment with FMD cycles improves memory in male 3xTg mice,
and reduces microglia activation in 3xTg mice. ........................................................................... 27
Figure 14. Short-term cycles of FMD regulate Nox2 cortex levels in 3xTg mice. ...................... 29
Figure 15. NOX2 deletion or inhibition improves cognitive behavior, mitigates pathology
progression and reduces microglia activation. ............................................................................. 34
Figure 16. Short-term cycles of FMD increase oligomeric Aβ42 internalization by IBA-1
positive microglia isolated from 8.5-month-old 3xTg mice. ........................................................ 35
Figure 17. Experimental timeline and percent survival in ASO and WT male mice. .................. 73
Figure 18. FMD cycles increase the forelimb steps in a behavior test of spontaneous activity
in a cylinder in male ASO and WT mice. ..................................................................................... 75
Figure 19. FMD cycles show a trend towards an increase in the hindlimb steps in a behavior
test of spontaneous activity in male ASO and WT mice. ............................................................ 77
Figure 20. FMD cycles do not increase the number of rears in a behavior test of
spontaneous activity in male ASO and WT mice. ....................................................................... 79
Figure 21. FMD cycles do not increase grooming time in a behavior test of spontaneous
activity in male ASO and WT mice. ............................................................................................. 80
Figure 22. Time to traverse across a challenging beam in male ASO and WT mice on either
FMD or a Control diet. .................................................................................................................. 82
Figure 23. Fine motor skills (nest building behavior) in 8.5 month old male ASO and WT
mice on either FMD or a Control diet. .......................................................................................... 83
Figure 24. FMD cycles do not significantly alter IGF-1 levels in male ASO and WT mice
at 9 months of age but reduce IGF-1 levels in WT and ASO groups compared to WT
control at 14.5 months of age. ....................................................................................................... 84
Figure 25. FMD cycles reduce the expression of mRNA levels of genes associated
with neuroinflammation and microglial activation (IL1β and CD68) in the mid-brain
from 14.5-month-old ASO mice. .................................................................................................. 86
Figure 26. Proteinase K resistant Alpha Synuclein Aggregates in the Substantia of male
ASO Control versus ASO FMD mice at 14.5 months of age. ..................................................... 87
ix
Abbreviations
Alzheimer’s Disease (AD)
Parkinson’s disease (PD)
Fasting Mimicking Diet (FMD)
Amyloid-β oligomers (oAβ)
Caloric restriction (CR)
Intermittent Fasting (IF)
Insulin-like Growth Factor 1 (IGF-1)
Protein kinase A (PKA)
Integrin subunit alpha M (CD11b)
Co-immunoprecipitation (co-IP)
NADPH oxidase (Nox)
lipopolysaccharide (LPS)
Amyloid precursor protein (APP)
4% Protein restriction (4% PR)
Alternate Day Fasting (ADF)
Spontaneous Alternation Behavior (SAB)
Escape box (EB)
Ionized calcium-binding adaptor protein-1 (Iba1)
Paraformaldehyde (PFA)
Immunohistochemistry (IHC)
Immunoreactivity (IR)
Peroxisome proliferator-activated receptor-γ (PPARγ)
x
Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α)
Lysosomal Autophagy System (LAS)
Mammalian target of rapamycin complex 1 (mTORC1)
AMP-dependent protein kinase (AMPK)
Uncoordinated-51 kinase (ULK1)
Phosphatidylinositol 3-phosphate (PI3P)
Phosphatidylethanolamine (PE)
Transcription factor EB (TFEB)
1- Methyl-1,2,3,6-tetrahydropyridine (MPTP)
6-Hydroxydopamine (6-OHDA)
Paraquat (PQ)
Leucine rich repeat kinase 2 gene (LRRK2)
PTEN-induced novel kinase 1 (PINK1)
Dietary restriction (DR)
2-Deoxy-D-glucose (2-DG)
Heat shock protein-70 (HSP-70)
Glucose regulated protein-78 (GRP-78)
Intermittent fasting (IF)
Substantia nigra (SN)
Inflammatory Bowel Disease (IBD)
Thy-1 Alpha Synuclein overexpressing (ASO)
Wild- type (WT)
Mouse on Mouse (MOM) blocking solution
xi
Normal Donkey Serum (NDS)
Normal Goat Serum (NGS)
Cluster of differentiation 68 (CD68)
Real-Time quantitative PCR (RT qPCR)
Interleukin-1β (IL1β)
xii
Abstract
A diet that mimics the effects of fasting without the adverse effects of water only
fasting has been shown to prevent or slow the progression of different disease such as diabetes,
cancer, multiple sclerosis and inflammatory bowel disease. Fasting Mimicking diet (FMD) also
extended longevity in mice administered the diet starting at middle age and reduced disease risk
factors for individuals from 20 to 70 years of age. Through the down regulation of Insulin-like
Growth Factor 1 (IGF-1) and Protein kinase A (PKA) signaling, FMD was found to promote
neurogenesis and enhance cognition as well as motor coordination in old mice. In a Multiple
Sclerosis model, FMD reduced inflammatory cells as well as pro inflammatory cytokines.
We studied the effects of FMD cycles in genetic mouse models of Alzheimer’s and
Parkinson’s disease. We found that FMD cycles improve cognition, ameliorate pathology,
increase neurogenesis and reduce neuroinflammation the 3xTg and E4FAD mouse model of
Alzheimer’s disease. FMD cycles also lowered oxidative stress marker, NADPH Oxidase (Nox2)
levels in 3xTg mice. In a genetic mouse model of Parkinson’s disease in which human Alpha
Synuclein is overexpressed, we found that long term cycles of FMD, improved moto co-
ordination, reduced the size of proteinase K resistant Alpha Synuclein aggregates in the
Substantia Nigra (SN) and reduced gene expression of neuroinflammation markers in the mid-
brain.
The results from these studies indicate that FMD is a potential treatment option for
neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease with its wide acting
effects on neuroinflammation, neurogenesis, behavior (cognitive as well as motor) and
ameliorating pathology in mouse models of these diseases.
1
Chapter 1: Dietary Interventions in Alzheimer’s Disease (AD)
1.1 Pathogenesis of Alzheimer’s disease
AD is a neurodegenerative disease that is characterized by the accumulation of amyloid
beta (oligomers and fibrils) (Gong et al. 2003) as well as hyperphosphorylated tau protein
(Bloom 2014). The neuropathology associated with AD can lead to inflammation, oxidative
damage, synaptic degeneration and neuronal death. As a result the learning and memory
functions of the cerebral cortex and hippocampus are affected(Cline et al. 2018).
Inflammation and oxidative stress can be damaging to neurons and also contribute to the
accumulation of Aβ in the brain (Block 2008). This leads to the activation of microglia, that
respond to the accumulation of amyloid proteins by changing morphology such as up-regulating
or synthetizing de novo surface receptors, secreting pro-inflammatory cytokines and reactive
species of oxygen (ROS). ROS include Nitric oxide and superoxide (Schlachetzki and Hüll
2009). The NADPH oxidase enzymatic complex (NOX2), that produced superoxide in microglia
cells, was proposed as a possible therapeutic target for the development of treatments for AD
(Block 2008). In the Longo lab, we have previously shown that Aβ can stimulate an increase in
superoxide production in neurons (Longo et al. 2000) along with the generation of microglial-
derived peroxynitrite that is highly toxic to neurons(Xie et al. 2002). It is also known that
peroxynitrite generated by inducible nitric oxide synthase (iNOS) and NADPH oxidase is a
mediator of microglial toxicity to oligodendrocytes (Li et al. 2005).
2
1.2 Current treatments for Alzheimer’s disease
The efficacy of drugs approved for Alzheimer’s disease has been limited (Connelly et al.
2019, Schneider et al. 2011). A recent effort to remove Aβ by an antibody-based intervention
was promising leading to improvements in cognition in AD patients. However, the high
incidence of amyloid-related imaging abnormalities (ARIA), especially in APOE4 carriers
treated with the higher and effective doses may limit its efficacy and safety for treatment of AD
(Plotkin and Cashman 2020). There is definitely a need for broader acting as well as safer
interventions.
1.3 Animal models of AD
Studies of multiple mouse models of AD can shed light on the molecular mechanisms
leading to AD and help to identify interventions to slow the progression of AD-associated
symptoms and pathology. The 3xTg-AD mouse model (3xTg) exhibits characteristic pathology
of human disease namely- Aβ and tau pathology (Oddo et al. 2003, Sterniczuk et al. 2010).On
the other hand, the EFAD mouse model (Youmans et al. 2012) was generated by having the
human APOE genotypes (APOE2, APOE3, APOE4) knocked into the 5xFAD-Tg mice(Oakley et
al. 2006) (Oakley et al., 2006). This mouse model allows for the investigation of the interactive
effects of APOE and AD pathology using E2FAD, E3FAD or E4FAD mice (Lewandowski,
Maldonado Weng and LaDu 2020).
3
1.4 Alzheimer’s disease and Dietary restriction
Previous research suggests that different forms of dietary interventions have potential
in slowing the progression of AD in mouse models. Caloric restriction (CR) generally refers to a
20% - 40% reduction in total calorie intake, while the levels of micronutrients are normal.
Dietary restriction (DR) refers to the restriction of a particular macronutrient (proteins, carbs or
fats) with or without a reduction in calorie intake(Mirzaei, Di Biase and Longo 2016).
Intermittent fasting (IF) includes daily fasting periods of 12 or more hours, twice weekly fasting
and alternate day fasting. Alternate day fasting is further defined as feeding every other day with
no or minimal food on the fasting day, along with no restriction on water intake (Longo and
Mattson 2014).
Calorie restriction (CR) studies conducted in PS1 mutant knock-in mice observed that an
alternate day fasting (IF) regimen of 3 months reduced excitotoxic damage to hippocampal CA1
and CA3 neurons compared to mice on a control diet (Zhu, Guo and Mattson 1999). Caloric
restriction for 14 weeks in APP and PS1 transgenic mice led to a reduction in Aβ pathology and
also decreased astrocyte activation that was associated with Aβ plaques (Patel et al. 2005). CR in
other AD mouse models also slowed the progression of Aβ deposition in the cerebral cortex and
hippocampal regions (Mouton et al. 2009, Halagappa et al. 2007). A previous study in 3xTg mice
undergoing a long term CR or intermittent fasting (IF) regimen for either 7 or 14 months indicated
that both CR and IF dietary regimens can ameliorate behavioral deficits by mechanisms that may
or may not be dependent on Aβ and tau pathologies(Halagappa et al. 2007). It is important to note
that chronic dietary restrictions are associated with both safety and compliance concerns,
especially in the older population that represents the majority of AD patients. In order to mitigate
these concerns, the Longo lab has investigated alternative interventions which may be feasible for
4
human testing. In human clinical trials, FMD cycles resulted in either no loss or an increase in lean
body mass as well as function (Wei et al. 2017, Caffa et al. 2020) . Our lab has previously showed
4 months of protein restriction cycles, alternated with normal feeding, improved behavior
performance in 3xTg male mice and reduced phosphorylated tau as compared to control mice.
(Parrella et al. 2013).
The Fasting Mimicking Diet (FMD) is composed of low calorie/low protein but high
unsaturated fat diet. FMD has an effect on stress resistance and longevity related markers similar
to the markers affected by fasting. However, it minimizes the burden of prolonged fasting as it
includes macro and micronutrients (Brandhorst et al. 2015). Based on our previous research that
the periodic use of a FMD can extend longevity and slow down cognitive decline in old wild type
mice (Brandhorst et al. 2015) and that protein restriction was able to improve cognition and reduce
hyperphosphorylated tau pathology in male 3xTg mice (Parrella et al. 2013), in this study we
investigated the effects of FMD cycles, administered in young male and female 3xTg and in female
E4FAD mice.
5
1.5 Hypothesis and Research Design
Our Hypothesis for this study is that an FMD regimen of 4-5 days followed by 9-10 days
of refeeding and repeated twice a month can slow the progression of Alzheimer’s disease in
mouse models through a reduction in neuroinflammation, oxidative stress and an increase in
regeneration. The latter would occur particularly during the refeeding phase.
We demonstrate that FMD cycles in 7-7.5-month-old female E4FAD mice, a) increased
spatial memory based on Barnes maze results b) reduced Aβ load in the cortex and hippocampus,
c) reduced expression of neurogenesis markers in the dentate gyrus (Sox2
+
and Ki67
+
), and d)
reduced NADPH Oxidase (Nox2) levels in the cortex. In ~18.5 month old male and female
3xTg mice, we found that FMD cycles, a) reduced Aβ load and hyperphosphorylated tau in the
hippocampus and b) reduced levels of microglia with modest shifts in their activation stages .
A short-term study of FMD starting at 6.5-month-old 3xTg mice for 2 months (5 cycles)
resulted in an improvement in short-term memory in male 3xTg mice by NOR, and a decrease in
microglial levels in female 3xTg mice. Nox2 levels were decreased in both male and female
3xTg mice when measured after 5 cycles of FMD, before refeeding. Results from 3xTg/Nox2-
KO and Apocynin treated 3xTg mice show that deletion or inhibition of Nox2 resulted in an
improvement in memory measured by NOR and Y-maze, decrease in microglia levels and a
reduction in hyperphosphorylated Tau (only in 3xTg/Nox2-KO mice).
In order to determine whether FMD cycles could slow the progression of AD pathology
and reduce the symptoms associated with it, male and female 3xTg mice were given FMD cycles
starting at 3.5 months till ~18.5 months of age and female E4FAD mice were administered the
diet at 3 months till ~7.5 months of age. The dietary intake and body weight of the mice were
monitored through the duration of the study.
6
To investigate further the mechanisms through which FMD cycles bring about changes in
3xTg mice, we conducted a short-term study, in which male and female 3xTg mice were
administered FMD cycles starting at 6.5 to 8.5 months of age and were euthanized after the 5
th
cycle, before refeeding. We also had a separate cohort of young male 3xTg mice that were given
FMD cycles starting at 6.5 months for a total of 4 cycles and were euthanized 2 days post
refeeding and female mice that were given 4 days of FMD (1 cycle) starting at around 8.5
months and were euthanized before refeeding.
End-point behavior (spatial memory) was measured in 6-6.5-month-old female E4FAD
mice as well as in the 18.5-month-old male and female 3xTg mice using the Barnes maze. In the
short term 3xTg study, short-term and working memory was tested using the NOR and Y-maze
respectively in 8.5-month-old male and female 3xTg mice.
The hippocampus of 18.5- month-old male and female 3xTg mice was stained for markers of
AD-related pathology such as Aβ and hyper-phosphorylated tau and CD11b and CD68 for
microglial activation/neuroinflammation. Aβ in the cortex and hippocampus, Ki67
+
and Sox2
+
cells in the dentate gyrus, cortex Nox2 levels and CD11b-Aβ42 interactions in the cortex were
assessed in female E4FAD mice. In our short-term FMD study of 3xTg mice, we evaluated IBA-
1 and Nox2 cortex levels before re-feeding (after 5 cycles of FMD in young male and female
3xTg mice and after 1 cycle in a separate cohort of young female 3xTg) and 2 days post-
refeeding (after 4 cycles of FMD in a separate cohort of young male 3xTg).
Thus, we have shown that FMD cycles have the potential to slow the progression of pathology
and symptoms related to Alzheimer’s disease, and can be neuroprotective or a safe and feasible
treatment for this neurodegenerative disease.
7
Chapter 2: The effects of a Fasting Mimicking Diet on cognition, pathology, neurogenesis,
neuroinflammation and NADPH Oxidase (Nox2) in E4FAD and 3XTGAD mouse models of
Alzheimer’s disease.
2.1 Abstract
Dietary interventions have the potential to offer neuroprotection as well as treat
neurodegenerative diseases. In this study we demonstrate that bi-monthly cycles of FMD in female
E4FAD mice for ~4 months starting at 3 months of age, resulted in an improvement in spatial
memory, reduced Amyloid Beta pathology, increased the number of proliferating neural stem cells
in the dentate gyrus and reduced NADPH Oxidase (Nox2) levels in the cortex.
We also observed that FMD cycles administered twice a month in male and female 3xTg
mice for ~15 months starting at 3.5 months of age, reduced AD-associated pathology (Amyloid
beta and hyperphosphorylated Tau) and reduced levels of microglia. Short term FMD cycles in
young 3xTg mice, improved short-term memory in male mice, reduced IBA-1 positive microglia
levels in female 3xTg mice and reduced the NADPH oxidase subunit, Nox2 in both male and
female mice after 5 cycles of FMD (prior to refeeding). Microglia isolated from the whole brain
(except cerebellum) of young 3xTg mice that were administered FMD for 5 cycles, 4 days post
refeeding, showed an increase in the ability to scavenge oligomeric Amyloid beta 42 in vitro.
Thus, our data indicate that FMD cycles can slow the progression of Alzheimer’s disease
pathology and ameliorate cognitive impairment in multiple mouse models through a modulation
of neuroinflammation, neurogenesis, and Nox2.
8
2.2 Results
FMD cycles improve spatial memory in female E4FAD mice.
In order to determine the effects of the FMD on mouse models of Alzheimer’s disease, we
first evaluated the effects of bi-monthly FMD cycles on cognition (spatial memory), Amyloid
beta pathology, neurogenesis and Nox2 in female E4FAD mice. The pathology in the cortex and
hippocampus is significantly increased by 6 months of age in female E4FAD mice that express
the human Apoe4 isoform (Youmans et al. 2012, Cacciottolo et al. 2016).
In our study, female E4FAD mice were assigned to either a Control group or an FMD
group starting at 3 months of age till ~7-7.5 months of age. The FMD mice were fed the diet for
5 days followed by 9 days of refeeding with a control diet (regular chow) and were given two
FMD cycles every month. The mice in the control group were administered regular chow for the
duration of the study. At the end of the FMD cycles, cognitive behavior was measured at ~6.5-7
months of age (Figure 1A).
Spatial memory was assessed using the Barnes maze in ~6.5-7-month-old E4FAD female
mice. During the 7-day training period as well as the retention test at day 14, FMD-treated mice
took significantly less time to find the escaped box (latency) (p<0.0001; Figure 1B) and were
more successful at finding the escape box in the given time (p<0.001; Figure 1C).
E4FAD mice on FMD cycles utilized more of the spatial strategy instead of the random
strategy to find the escape box compared to mice on the control diet (Figure 1D and 1E). This
data suggests that bi-monthly FMD cycles improve spatial memory in E4FAD female mice.
9
Figure 1. FMD cycles improve spatial memory in female E4FAD mice.
(A) Experimental timeline for female E4FAD mice starting at 2.5 months of age through 7-7.5 months of age.
(B) Latency between E4FAD FMD females (n=20) and E4FAD Control females (n=19) in the Barnes maze at approximately 6.5-
7 months.
(C) Success rate in finding the escape box between E4FAD FMD females (n=20) and E4FAD Control females (n=19) in the
Barnes maze at approximately 6.5-7 months.
(D) Strategies (random, serial, and spatial) employed by female E4FAD Control mice (n=19) to locate escape box.
(E) Strategies (random, serial, and spatial) employed by female E4FAD FMD mice (n=20) to locate escape box.
Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 and ****p<0.0001, 2-way ANOVA.
1 2 3 4 5 6 7 14
0
20
40
60
80
100
Days
Strategy Used to Find
Escape Box (%)
Random
Serial
Spatial
E4FAD Females
FMD
Barnes Maze Strategy - 6.5 Months
A
0 1 2 3 4 5 6 7 14
0
30
60
90
120
Days
Time Elapsed Before
Finding Escape Box (sec.)
Control Diet
FMD
E4FAD Females
****p<0.0001
Barnes Maze Latency - 6.5 Months
0 1 2 3 4 5 6 7 14
0
25
50
75
100
Days
Success in Finding
Escape Box (%)
Control Diet
FMD
E4FAD Females ***p<0.001
Barnes Maze Success - 6.5 Months
1 2 3 4 5 6 7 14
0
20
40
60
80
100
Days
Strategy Used to Find
Escape Box (%)
Random
Serial
Spatial
E4FAD Females
Control Diet
Barnes Maze Strategy - 6.5 Months
B C
E D
10
FMD cycles reduce hippocampal and cortex Aβ load, in female E4FAD mice.
Using Immunohistochemistry (IHC) approaches, we assessed Aβ immunoreactivity in the
hippocampus (Subiculum and CA1) and cortex of 7-7.5-month-old female mice on control or
FMD after ~4 months on the diet (Figure 2A and 8A). In comparison with E4FAD Control mice,
E4FAD FMD female mice reduced Aβ load in the subiculum (p<0.01; Figure 2B) and cortex,
(p<0.001; Figure 2D), but not in the CA1 region of the hippocampus (Figure 2C).
Figure 2. FMD cycles reduce hippocampal and cortex Aβ load, in female E4FAD mice.
(A) Representative images showing Aβ immunoreactivity in subiculum and cortex regions of female E4FAD Control and FMD
groups.
(B) Quantification of subiculum Aβ load (%) for female E4FAD Control (n=18) and FMD (n=18) groups.
(C) Quantification of CA1 Aβ load (%) for female E4FAD Control (n=18) and FMD (n=18) groups.
(D) Quantification of cortex Aβ load (%) for female E4FAD Control (n=17) and FMD (n=17) groups.
Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 and ***p < 0.001, Unpaired 2-tailed student’s t-test. Images were taken
at 20x magnification.
11
The effects of FMD cycles on neurogenesis and the number of astrocytes in the dentate gyrus
of female E4FAD mice.
We assessed markers of neurogenesis in ~7-7.5-month-old female E4FAD mice by
measuring cells that were Ki67
positive
in the dentate gyrus (Khuu et al. 2019, Ma et al. 2014,
Kee et al. 2002). We also quantified the cells that expressed Ki67 along with Sox2 which is a
neural stem cell marker in E4FAD control and FMD mice (Figure 3A). The number of Sox2
+
cells were not significantly different between the control and FMD groups (Figure 3B and 3E).
However, there was a significant increase in Ki67
+
Sox2
+
cells in mice FMD mice compared to
the mice in the control group (p<0.05; Figure 3C). This indicated an increase in neurogenesis in
female E4FAD mice administered FMD cycles for ~ 4 months.
To exclude the possibility that the Sox2
+
cells may be proliferating astrocytes,
immunofluorescence staining was carried out on sections from E4FAD control and FMD mice
using Sox2
+
and GFAP
+
antibodies. There was no significant difference in the number of Sox2
+
cells, GFAP
+
Sox2
+
cells and the percentage of GFAP
+
Sox2
+
cells / total Sox2
+
cells in the
dentate gyrus between the E4FAD control (n=3) and FMD (n=4) groups (Figure 3E, 3G, and
3H). However, there was a non-significant trend for an increase in the number of GFAP
+
cells in
the dentate gyrus in the E4FAD FMD mice compared to the E4FAD control mice (p=0.0715;
Figure 3F).
12
Figure 3. The effects of FMD cycles on neurogenesis and the number of astrocytes in the
dentate gyrus of female E4FAD mice.
(A)Representative images showing Sox2
+
, Ki67
+
, and co-stain hippocampal immunohistochemistry for ~7-7.5-month-old female
E4FAD Control and FMD groups. White arrows indicate the Ki67
+
/Sox2
+
foci.
(B) Quantification of ~7-7.5-month-old E4FAD female Sox2
+
[Control (n=13) and FMD (n=11)] cells in the dentate gyrus (DG)
after ~4 months of FMD cycles.
(C) Quantification of ~7-7.5-month-old E4FAD female Ki67
+
Sox2
+
[Control (n=13) and FMD (n=11)] cells in the DG after ~4
months of FMD cycles.
(D)Representative images showing Sox2
+
, GFAP
+
, and GFAP
+
Sox2
+
cells in the
subgranular zone (SGZ) of the dentate gyrus for ~7-7.5-month-old female E4FAD Control and FMD groups.
(E) Quantification of ~7-7.5-month-old E4FAD female Sox2
+
cells [Control (n=3) and FMD (n=4)] in the dentate gyrus (DG) after
~4 months of FMD cycles.
(F) Quantification of ~7-7.5-month-old E4FAD female GFAP
+
cells [Control (n=3) and FMD (n=4)] in the dentate gyrus (DG)
after ~4 months of FMD cycles.
(G) Quantification of ~7-7.5-month-old E4FAD female GFAP
+
Sox2
+
cells [Control (n=3) and FMD (n=4)] in the dentate gyrus
(DG) after ~4 months of FMD cycles.
(H) Quantification of the percentage of GFAP
+
Sox2
+
cells / total Sox2
+
cells in ~7-7.5-month-old E4FAD female mice [Control
(n=3) and FMD (n=4)] in the dentate gyrus (DG) after ~4 months of FMD cycles.
Data are presented as mean ± SEM. Unpaired 2-tailed student’s t-test. Images were taken at 20x magnification.
A
E F
0
20
40
60
80
Sox2
+
cells
E4FAD Control
E4FAD FMD
0
5
10
15
20
25
Ki67
+
Sox2
+
cells
*
E4FAD Control
E4FAD FMD
B C
D
Sox2
+
Ki67
+
FMD Control Diet
Merge
Sox2
+
GFAP
+
FMD Control Diet
Merge
0
5
10
15
20
25
GFAP
+
cells
E4FAD Control
E4FAD FMD
p=0.0715
G H
0
2
4
6
8
GFAP
+
Sox2
+
cells
E4FAD Control
E4FAD FMD
0
10
20
30
40
Sox2
+
cells
E4FAD Control
E4FAD FMD
0
10
20
30
40
Percentage of GFAP
+
Sox2
+
/ total Sox2
+
cells
E4FAD Control
E4FAD FMD
13
Assessment of CD11b-Aβ42 binding in detergent-soluble/triton-soluble cortex of female ~7-
7.5-month-old E4FAD mice.
Integrin subunit alpha M (CD11b) was previously shown to facilitate the clearance of Aβ
in AD patients (Zabel et al. 2013).We investigated whether there could be an interaction between
CD11b and Aβ in triton soluble extracts of the cortex from E4FAD mice at ~7-7.5 months of
age. There was no significant difference in Aβ42 fold-change between the E4FAD Control and
FMD E4FAD groups in our co-immunoprecipitation (co-IP analysis) of CD11b and Aβ42 binding
(Figure 4A and 4B).
Figure 4. Assessment of CD11b-Aβ42 binding in detergent-soluble/triton-soluble cortex of
female ~7-7.5-month-old E4FAD mice.
(A)Western blot image used for quantifying Aβ 42 fold change in detergent-soluble/triton-soluble cortex extract of Control (n=4)
and FMD (n=4) ~7-7.5-month-old female E4FAD mice.
(B) Co-immunoprecipitation (co-IP) of CD11b and Aβ 42 binding in detergent-soluble/triton-soluble cortex extract of Control
(n=4) and FMD (n=4) ~7-7.5-month-old female E4FAD mice (measured as Aβ 42 fold-change).
Data are presented as mean ± SEM. Unpaired 2-tailed student’s t-test.
FMD cycles reduce Nox2 levels in E4FAD mice.
Activated microglia have been known to promote neurotoxicity although microglia also have a
number of positive functions in the brain (Xie et al. 2002, Wilkinson and Landreth 2006, Richard
et al. 2008).
B A
0.0
0.5
1.0
1.5
2.0
Cortex Aβ
42
fold-change
E4FAD Females
Control
Diet
FMD
14
The activation of NADPH oxidase (Nox) can result in the production of superoxide (O2
-
)
by the microglia. Microglial superoxide can contribute to the development of AD and other
neurological diseases(Babior 2000, Chéret et al. 2008, Harrigan et al. 2008, Mander and Brown
2005, Sankarapandi et al. 1998, Shimohama et al. 2000, Wilkinson and Landreth 2006, Zekry,
Epperson and Krause 2003). Previous work in the Longo lab has shown that Superoxide along
with iron can contribute to neurotoxicity in Aβ-treated neuronal cell lines (Longo et al. 2000).
We have also shown previously that peroxynitrite (ONOO
-
) that is formed by the reaction
between Nitric Oxide (NO) and Superoxide (O2
-
), mediates the neurotoxicity promoted by Aβ or
lipopolysaccharide (LPS) activated microglia in vitro (Xie et al. 2002). Nox2 is a key enzyme
involved in O2
-
production and targeting it has been shown to reduce neurotoxicity in vitro (Qin
et al. 2002, Qin et al. 2006) . In mice overexpressing the Swedish mutation of the amyloid
precursor protein (APP) lacking the catalytic subunit of Nox2, showed a reduction in
cerebrovascular and cognitive dysfunctions (Park et al. 2008).
We assessed whether the levels of Nox2, were affected at the end of ~4 months of FMD
cycles in female E4FAD mice and observed a non-significant trend towards a reduction of Nox2
levels after re-feeding in FMD-treated female E4FAD mice (p=0.0549; Figure 5A and 5B).
0.0
0.1
0.2
0.3
0.4
0.5
Nox2 /Vinculin expression (Cortex)
p=0.0549
E4FAD
Control
E4FAD
FMD
A B
15
Figure 5. FMD cycles reduce Nox2 levels in E4FAD mice.
(A)Quantification of Nox2 levels in hemi cortex extract of Control (n=5) and FMD (n=4) ~7-7.5-month-old female E4FAD mice
after 4 months of bi-weekly FMD cycles (measured as Nox2/Vinculin protein expression levels).
(B) Western blot image used for quantifying Nox2 levels in hemi-cortex extract of Control (n=5) and FMD (n=4) ~7-7.5-month-
old female E4FAD mice. Vinculin was loading control (bottom).
Data are presented as mean ± SEM. Unpaired 2-tailed student’s t-test.
Body weight profiles of 18.5-month-old 3xTg-AD male and female mice after the first four
weeks, at the mid-point and endpoint of the long-term dietary treatment.
We also administered FMD cycles to 3xTg male and female mice starting at 3.5 months
of age till 18.5 months of age. In addition to a group on a standard rodent chow diet (Control), and
a group on a FMD for 4-5 days (FMD; 4 days for males and 5 days for females) we studied a group
that consumed a 7-day protein-restricted diet, [approximately 4% protein in composition relative
to carbohydrates and fat (4% PR)], based on our previous studies indicating that alternate weeks
of protein/essential amino acids restriction mediates part of the effects of FMDs (Parrella et al.
2013, Levine et al. 2014). FMD males underwent a 4-day cycle vs. a 5-day cycle, since 3xTg males
were found to lose weight more rapidly and to a higher extent as compared to female mice. 3xTg
male and female Control mice weighed an average of 40.3g and 34.7g respectively at 18.5 months.
3xTg male and female mice in the FMD and 4% PR groups regained most of their weight lost
during the diet cycles upon refeeding (Figure 6 A-6F).
16
Figure 6. Body weight profiles of 18.5-month-old 3xTg-AD male and female mice after the
first four weeks, at the mid-point and end-point of the long term dietary treatment.
(A) Body weight profile for 3xTg females in Control (n=10), FMD (n=17) and 4% PR (n=16), groups after the first four weeks of
diet.
(B) Body weight profile for 3xTg males in Control (n=9), FMD (n=19) and 4% PR (n=16), groups after the first four weeks of
diet.
(C) Body weight profile for 3xTg females in Control (n=10), FMD (n=16) and 4% PR (n=16) groups at mid-point (~10.5-months
old).
(D) Body weight profile for 3xTg males in Control (n=8), FMD (n=17) and 4% PR (n=16) groups at mid-point (~10.5-months old).
(E) Body weight profile for 3xTg males in Control (n=9), FMD (n=16) and 4% PR (n=14), groups at endpoint (~18.5-months old).
(F) Body weight profile for 3xTg males in Control (n=8), FMD (n=16) and 4% PR (n=16), groups at endpoint (~18.5-months old).
B
C D
E
A
F
3xTg Females- Body weight (First four weeks)
0 5 10 15 20 25 30
0
10
20
30
40
Days
Body weight (g)
Control (n=10)
FMD (n=17)
4% PR (n=16)
3xTg Females- Body weight (Mid-point)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
10
20
30
40
Days
Body weight (g)
Control (n=10)
FMD (n=16)
4% PR (n=16)
3xTg Males- Body weight (End-point)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
10
20
30
40
50
Days
Body weight (g)
FMD (n=16)
Control (n=8)
4% PR (n=16)
3xTg Females- Body weight (End-point)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
10
20
30
40
Days
Body weight (g)
Control
FMD
4% PR
3xTg Males- Body weight (First four weeks)
0 5 10 15 20 25 30
0
10
20
30
40
50
Days
Body weight (g)
FMD
Control
4% PR
3xTg Males- Body weight (Mid-point)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
10
20
30
40
50
Days
Body weight (g)
FMD
Control
4% PR
17
FMD cycles slow the progression of AD-associated pathology in aged 3xTg mice.
Aβ accumulation and hyper-phosphorylated tau are well-established markers for AD (Lo
et al. 2013). Hence, we assessed hippocampal Aβ load and the number of hippocampal neurons
that are AT8
+
, a marker for abnormally phosphorylated tau(Parrella et al. 2013), in female and
male 3xTg mice at the end of the study. We stained for Aβ in the subiculum and CA1
hippocampal regions after ~15 months of dietary regimens in approximately 18.5-month-old
female 3xTg mice (Figure 7A, top and middle; Figure 8B, top). We observed that female 3xTg
mice treated with the periodic FMD had a reduced Aβ load in the subiculum (p<0.01, Figure 7B)
and in CA1 regions (p<0.05, Figure 7C) as compared to Control 3xTg mice. A similar reduction
in Aβ load was observed in the subiculum of 4% PR female 3xTg mice compared with the
Control 3xTg females (p<0.01, Figure 7B). The number of AT8
+
neurons in the combined
subiculum and CA1 regions from female 3xTg groups (Figure 7A, bottom; Figure 8B, bottom)
was reduced in FMD-treated 3xTg females as compared to the control 3xTg mice (p<0.01,
Figure 7D), while there was no significant difference in AT8
+
neuron number between the 4%
PR 3xTg and the 3xTg control animals (Figure 7D). In the subiculum and CA1 hippocampal
regions of aged male 3xTg mice after ~15 months of dietary regimens (18.5 months of age)
(Figure 7E, top and middle; Figure 8C, top), there were no significant reductions in subiculum
(Figure 7F) or CA1 (Figure 7G) Aβ load between the FMD and control 3xTg groups. A non-
significant trend towards a reduction in Aβ load in the CA1 was observed in the 4% PR male
3xTg group as compared with the Control 3xTg males (p=0.0525, Figure 7G). Concerning
hyper-phosphorylated tau counts, the number of AT8
+
neurons in the combined subiculum and
CA1 regions for the male 3xTg groups (Figure 7E, bottom ; Figure 8C, bottom) was reduced in
the FMD 3xTg mice compared to the 3xTg controls (p<0.05, Figure 7H). A similar reduction in
18
the number of AT8
+
neurons was observed in the 4% PR animals and FMD group as compared
with the 3xTg Controls (Figure 7E, bottom ; Figure 8C, bottom) (p<0.05, Figure 7H). These
results indicate that FMD cycles reduce both Aβ load or hyper-phosphorylated tau in female and
male mice, in part through the temporary reduction of protein intake.
Figure 7. FMD cycles slow the progression of AD-associated pathology in aged 3xTg mice.
(A) Representative images showing Aβ immunoreactivity and AT8-positive neurons (recognizes abnormally phosphorylated tau)
in subiculum or CA1 hippocampus regions of female Control, FMD and 4% PR 3xTg mice.
(B) Quantification of subiculum Aβ load (%) for female 3xTg Control (n=21), FMD (n=16), 4% PR (n=14) mice.
(C) Quantification of CA1 Aβ load (%) for female 3xTg Control (n= 20), FMD (n=16) and 4% PR (n=14) mice.
(D) Quantification of AT8
+
neurons in the subiculum and CA1 for female 3xTg Control (n=20), FMD (n=14) and PR (n=12)
mice.
(E) Representative images showing Aβ immunoreactivity and AT8
+
neurons in subiculum or CA1 hippocampus regions of male
3xTg Control, FMD and 4% PR mice.
(F) Quantification of subiculum Aβ load (%) for male 3xTg Control (n=15), FMD (n=14) and 4%PR (n=12) mice.
(G) Quantification of CA1 Aβ load (%) for male 3xTg Control (n=15), FMD (n=14) and 4%PR (n=12) mice.
(H) Quantification of AT8
+
neurons in the subiculum and CA1 for male 3xTg Control (n=12), FMD (n=13) and 4%PR (n=11)
mice.
Data are presented as mean ± SEM.
For Figure 7B-7D, 7F-7H; ∗p < 0.05 and ∗∗p < 0.01, one-way ANOVA.
Images were taken at 20x magnification.
Control
FMD
4% PR
0
100
200
300
400
500
AT8
+
Cells
**
Control
FMD
4% PR
0
5
10
15
20
CA1 Aß Load (%)
*
Control
FMD
4% PR
0
75
150
225
300
375
AT8
+
Cells
*
*
A
B C D
3xTg Females
E
F G H
3xTg Males
Control
FMD
4% PR
0
10
20
30
40
50
Aß Load (Subiculum) (%)
**
**
Control
FMD
4% PR
0
10
20
30
40
Aß Load (Subiculum) (%)
Control
FMD
4% PR
0
5
10
15
20
25
CA1 Aß Load (%)
p=0.0525
19
Figure 8. FMD cycles slow the progression of AD-associated pathology in E4FAD, aged and
8.5-month-old 3xTg mice. Related to Figure 2 and 7.
(A) Representative images showing Aβ immunoreactivity in subiculum and cortex regions of female 7-7.5-month-old E4FAD
Control and FMD groups.
(B) Representative images showing subiculum and CA1 Aβ immunoreactivity and AT8
+
neurons in hippocampus for 18.5-
month-old 3xTg female Control, FMD and 4% PR groups.
(C) Representative images showing subiculum and CA1 Aβ immunoreactivity and AT8
+
neurons in hippocampus for 18.5-
month-old 3xTg male Control, FMD and 4% PR groups.
FMD cycles regulate CD11b-immunoreactive (CD11b-ir) and CD68 positive microglia
levels in aged 3xTg mice.
To assess potential improvements in neuroinflammation, we stained for CD11b-activated
microglia in the hippocampi of 18.5-month-old wildtype male and female mice and compared
them to the Control and FMD 18.5-month-old 3xTg female and male groups (Figure 9A and
9D). Among females, there was a major increase in microglia density in the combined subiculum
and CA1 regions of the 3xTg mice on a standard diet as compared to microglia density in
B C
D E
A
4% PR
18.5 mo old 3xTg Females
Control
FMD
Amyloid β
Tau
Amyloid β
4% PR
18.5 mo old 3xTg Males
Control FMD
Tau
Amyloid β
E4FAD Females
Control FMD
8.5 mo old 3xTg Males
Control FMD
Tau
Amyloid β
8.5 mo old 3xTg Females
Control FMD
Tau
Amyloid β
20
wildtype controls (p<0.01; Figure 9B). FMD cycles reduced microglia to a level that was no
longer significantly higher than that of the wild type mice (Figure 9B). Female 3xTg mice that
were subjected either to the Control diet or to the periodic FMD showed a lower proportion of
resting state microglia and a higher proportion of activated and amoeboid microglia when
compared with the age-matched wildtype mice (stage 3: WT vs. Control, *p<0.05; WT vs. FMD,
**p<0.01; Figure 9C). Similarly, among male mice, there was a major increase in microglia
density in the combined subiculum and CA1 regions of the 3xTg mice on a standard diet as
compared to that in wildtype controls (p<0.05; Figure 9E). FMD cycles reduced microglia to a
level that was no longer significantly higher than that of the wild type mice (Figure 9E). When
microglia activation stages were examined, the 3xTg mice on the control or FMD diets displayed
either a lower or a trend towards a lower proportion of resting state microglia and a higher
proportion of amoeboid microglia as compared to the age-matched wild type mice on the
standard diet (stage 1: WT vs. Control ,*p<0.05; stage 3 : WT vs. Control, ***p<0.0001; WT vs.
FMD,*p<0.05; stage 4: WT vs. Control and WT vs. FMD, *p<0.05; Figure 9F). These results
indicate that FMD cycles cause a reduction in the number of CD11b-labeled microglia but do not
affect their activation state. FMD cycles also reduced the average number of Cluster of
differentiation 68 positive (CD68) microglia in the hippocampus of male 3xTg mice (p<0.05;
Figure 10A-B).
Overall, these data indicate that a long-term regimen of FMD cycles can reduce AD-
associated pathology in aged male and female 3xTg mice, possibly by reducing microglia density
and by modulating microglia activation state.
21
Figure 9. FMD cycles regulate CD11b-immunoreactive (CD11b-ir) microglia levels and
activation in aged 3xTg mice.
(A) Representative images showing CD11b-ir microglia in hippocampus sections of 18.5-month-old female C57B/6 wildtype,
3xTg Control and 3xTg FMD mice .
(B) Quantification of density of CD11b-ir cells in hippocampus CA1 and subiculum combined brain regions of female C57B/6
wildtype, 3xTg Control, and 3xTg FMD groups (n=5-7 animals/group).
(C)Percentage of different microglia activation stages (from 1 to 4) of female C57B/6 wildtype, 3xTg Control and 3xTg FMD
mice ( n=5-7 animals/group).
(D) Representative images showing CD11b-ir microglia in hippocampus sections of 18.5-month-old male C57B/6 wildtype,
3xTg Control and 3xTg FMD mice.
(E) Quantification of density of CD11b-ir cells in hippocampus CA1 and subiculum combined brain regions of male C57B/6
wildtype, 3xTg Control and 3xTg FMD (n=5-8 animals/group)
(F) Percentage of different microglia activation stages (from 1 to 4) of male C57B/6 wildtype, 3xTg Control and 3xTg FMD
mice (bottom right; n=5-8 animals/group).
Data are presented as mean ± SEM.
∗p < 0.05 and ∗∗p < 0.01, one-way ANOVA.
Images were taken at 20x magnification.
Figure 10. FMD regulate CD68 positive microglia in aged 3xTg mice.
(A) Representative images showing CD68+ microglia in hippocampus sections of 18.5-month-old male 3xTg Control and 3xTg
FMD mice (n=2-3 animals/group).
(B) Quantification of density of CD68+ cells in hippocampus CA1 and subiculum combined brain regions of male 3xTg Control
and 3xTg FMD mice (n=2-3 animals/group).
Data are presented as mean ± SEM.
∗p < 0.05, Unpaired 2-tailed student’s t-test.
Images were taken at 20x magnification.
A
B C
D
3xTg Females
E F
3xTg Males
resting activated amoeboid phagocytic resting activated amoeboid phagocytic
3xTg Males
3xTg Control
3xTg FMD
0
50
100
150
200
250
Average # of Microglia (CD68+)
Per Total Subiculum and CA1 Area
*
A
3xTg Control 3xTg FMD
CD68
Immunoreactivity
B
22
FMD cycles do not improve spatial memory in female and male 3xTg mice assessed at 17.5-
18 months of age.
At the end of the long-term FMD study in 3xTg mice, a behavioral assay for spatial
memory was conducted when the mice were ~ 18 months of age (Figure 11 A-G and 12 A-G).
Both male and female 18-month-old 3xTg mice after ~14.5 months of FMD and 4% PR dietary
regimens as well as age-matched controls were tested in the Barnes maze. Of the various
combinations tested, a non-significant trend for the female 3xTg treated with the FMD was
observed in terms of a reduction in latency (time spent to locate escape box) compared to female
3xTg controls (Figure 11D). The female 3xTg FMD group also had a non-significant trend for an
increased success rate in finding the escape box compared to the 3xTg female controls (Figure
11E).
Although there was a reduction in Amyloid and Tau pathology in old 3xTg mice, the
accumulation of pathology appears to minimize the effects of the FMD in improving cognition in
18.5-month-old 3xTg mice.
23
Figure 11. FMD cycles do not improve spatial memory in female 3xTg mice assessed at
17.5-18 months of age.
(A) Experimental diet and behavior schedule for female FMD and 3xTg control mice starting at 3.5 months of age through 18.5
months of age.
(B) Number of errors between 3xTg FMD females (n=14) and 3xTg females on control diet (n=19) in the Barnes maze at
approximately 17.5-18 months.
(C) Deviation (number of the holes from the escape box in which the mouse makes its first error) between 3xTg FMD females
(n=14) and 3xTg females on control diet (n=19) in the Barnes maze at approximately 17.5-18 months.
(D) Latency (seconds lapsed before finding escape box) between 3xTg FMD females (n=14) and 3xTg females on control diet
(n=19) in the Barnes maze at approximately 17.5-18 months.
(E) Success rate in finding the escape box between 3xTg FMD females (n=14) and 3xTg females on control diet (n=19) in the
Barnes maze at approximately 17.5-18 months.
(F) Strategies (random, serial, and spatial) used by female 3xTg Control group (n=19) to locate escape box.
(G) Strategies (random, serial, and spatial) used by female 3xTgFMD group (n=14) to locate escape box.
Data are presented as mean ± SEM.
24
Figure 12. FMD cycles do not improve spatial memory in male 3xTg mice assessed at 17.5-
18 months of age.
(A) Experimental diet and behavior schedule for male FMD and 3xTg control mice starting at 3.5 months of age through 18.5
months of age.
(B) Number of errors between 3xTg FMD males (n=11) and 3xTg males on control diet (n=11) in the Barnes maze at
approximately 17.5-18 months.
(C) Deviation (number of the holes from the escape box in which the mouse makes its first error) between 3xTg FMD males
(n=11) and 3xTg males on control diet (n=11) in the Barnes maze at approximately 17.5-18 months.
(D) Latency (seconds lapsed before finding escape box) between 3xTg FMD males (n=11) and 3xTg males on control diet (n=11)
in the Barnes maze at approximately 17.5-18 months. (E) Success rate in finding the escape box between 3xTg FMD males
(n=11) and 3xTg males on control diet (n=11) in the Barnes maze at approximately 17.5-18 months.
(F) Strategies (random, serial, and spatial) used by male 3xTg Control group (n=11) to locate escape box.
(G) Strategies (random, serial, and spatial) used by male 3xTg FMD group (n=11) to locate escape box.
Data are presented as mean ± SEM. ∗p < 0.05, two-way ANOVA.
25
Short- term treatment with FMD cycles improves memory in male 3xTg mice and reduces
microglia activation in 3xTg mice.
To investigate further the mechanisms responsible for the effects of long-term dietary
treatment of 3xTg mice we administered FMD cycles over a short-term period to mice at an age
when AD pathology is increasing rapidly and begins to influence cognitive behavior and
microglia activation in the brain. Specifically, we treated male and female 3xTg mice at
approximately 6.5 months of age with either a Control diet or FMD cycles (Figure 13A). At this
stage, intraneuronal and extracellular Aβ were previously found to begin to develop in the
hippocampus and in the cortex, and tau pathology is in its nascent stages (Oddo et al. 2003).
Male and female 3xTg mice from the FMD group were administered a 4-day FMD, for 5 cycles
(4 days diet, 10 days re-feeding), and were sacrificed after the 5
th
cycle, and before refeeding.
Cognitive behavior was assessed after 4 cycles, when mice were approximately 8.5
months old and before the last and 5
th
cycle. SAB results showed no significant differences
among the male or female cohorts (Figure 13D and 13E). However, 3xTg mice did not display a
reduction in SAB performance compared to wildtype controls, suggesting that at this age, AD
mice did not develop significant dysfunction. NOR results instead suggest the 3xTg FMD males
had a significantly higher average RI score as compared to that in 3xTg Control male mice
(p<0.05, Figure 13C). Again, 3xTg mice did not display a reduction in NOR performance
compared to wildtype controls, suggesting that the effect of the FMD may represent an
improvement even compared to wildtype control mice and in agreement with our previous
studies on FMD in wildtype mice (Brandhorst et al. 2015). No significant differences were
observed among the female groups (Figure 13B).
We stained hippocampal tissue for ionized calcium-binding adaptor protein-1 (Iba1),
counted Iba1 density, and categorized the stages of microglial activation based on criteria
26
established in previous studies (Kreutzberg 1996, Crews and Vetreno 2016)(Figure 13 F and
13G, top), with the intent of observing possible changes that could impact neuroinflammation in
these relatively young mice. 3xTg female mice displayed a major increase in microglial number
compared to wildtype controls (p<0.0001, Figure 13F, bottom left) which was reduced in 3xTg
FMD females (p<0.001, Figure 13F, bottom left). There was a significant reduction in microglia
at stage 1 of activation in the 3xTg female controls as compared to wildtype (p<0.05, Figure 13F,
bottom right). Microglia at stage 3 and 4 of activation was significantly increased in the 3xTg
Control group compared to wildtype (stage 3: p<0.01, stage 4: p<0.05, Figure 13F, bottom right).
3xTg FMD females displayed a non-significant trend for the reduction in microglia at stage 4
compared to the 3xTg Control group (p=0.0772, Figure 13F, bottom right). Among the groups
in the male cohort, there was no significant increase in microglia number among any of the male
groups (Figure 13G, bottom left). Shifts in activation stages were apparent, with both 3xTg male
groups having significantly less microglia at stage 1 compared to wild type males (p<0.05,
Figure 13G, bottom right). The 3xTg control group displayed a trend for an increase in microglia
at stage 3 compared to wildtype males (p=0.0652, Figure 13G, bottom right) and for an increase
in microglia at stage 4 of activation as compared to wildtype males and 3xTg FMD males
(Figure 13G, bottom right). In summary, as observed for 18-month-old mice, microglial levels
were increased in 3xTg mice and this increase was partially reversed in FMD-treated mice. In
contrast, resting state microglia was reduced and different forms of activated microglia were
increased in 3xTg as compared to wild type mice. In this set of short-term treatment experiments,
the FMD appears to allow a general state of microglial activation while reducing the highly
active phagocytic microglia in the 3xTg FMD-treated mice.
27
Figure 13. Short- term treatment with FMD cycles improves memory in male 3xTg mice,
and reduces microglia activation in 3xTg mice.
(A) Experimental diet and behavior schedule for 3xTg males and females starting at 6.5 months of age through approximately 8.5
months of age for 5 FMD cycles.
(B) RI for trial 2 of NOR task for 8.5-month-old C57B/6 wildtype males (n=8) and 8.5-month-old 3xTg male Control (n=10) and
FMD after 4 cycles of FMD and 7 days refeeding (n=5).
(C) RI for trial 2 of NOR task for C57B/6 wildtype females (n=7) and 3xTg female Control (n=7) and FMD (n=8) groups.
(D) SAB percentage for C57B/6 wildtype females (n=8) and 3xTg female Control (n=9) and FMD groups (n=9).
(E) SAB percentage for C57B/6 wildtype males (n=8) and 3xTg male Control (n=10) and FMD (n=7) groups.
(F) Representative images showing Iba1 stained microglia in hippocampus sections of 8.5-month-old female C57B/6 wildtype
and 3xTg Control and FMD groups (top). Quantification of density of Iba1
+
microglia in the CA1 and subiculum hippocampus
regions of C57B/6 wildtype females (n=8) and 3xTg female Control (n=7) and FMD (n=7) groups (bottom left). Percentage of
different microglia activation stages (from 1, resting to 4, most activated) of C57B/6 wildtype females (n=8) and 3xTg female
Control (n=7) and FMD (n=7) groups (bottom right).
(G) Representative images showing Iba1 stained microglia in hippocampus sections of 8.5-month-old male C57B/6 wildtype and
3xTg Control and FMD groups (top). Quantification of density of Iba1
+
microglia in the CA1 and subiculum hippocampus
regions of C57B/6 wildtype males (n=8) and 3xTg male Control (n=16) and FMD (n=5) groups (bottom left). Percentage of
different microglia activation stages (from 1, resting to 4, most activated) of C57B/6 wildtype males (n=8) and 3xTg male
Control (n=16) and FMD (n=5) groups (bottom right).
Data are presented as mean ± SEM.
For Figure 13B-E: *p < 0.05, compared with WT; one-way ANOVA followed by Tukey’s multiple comparisons test.
For Figure 13F and 13G: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; one-way ANOVA followed by Tukey’s multiple
comparisons test.
Images were taken at 20x magnification unless otherwise noted.
Wildtype
3xTg Control
3xTg FMD
0
20
40
60
80
100
Recognition Index (Novel/Both;sec)
*
Males - 8.5 mos. - NOR
B
Wildtype
3xTg Control
3xTg FMD
0
100
200
300
Average # of Microglia (Iba1
+
)
Per Total Subiculum and CA1 Area
n.s.
Males - 8.5 mos.
Wildtype
3xTg Control
3xTg FMD
0
100
200
300
400
Average # of Microglia (Iba1
+
)
Per Total Subiculum and CA1 Area
***
****
****
Females - 8.5 mos.
0
20
40
60
80
Microglia
activation stages (%)
1 2 3 4
3xTg Control
3xTg FMD
Wildtype
Significance compared to WT (*)
* *
p=0.0652
Males - 8.5 mos.
resting activated amoeboid phagocytic
0
20
40
60
80
Microglia
activation stages (%)
1 2 3 4
Wildtype
3xTg Control
3xTg FMD
**
*
p=0.0772
Females - 8.5 mos.
Significance compared to WT (*)
*
resting activated amoeboid phagocytic
C
F
A
Wildtype
3xTg Control
3xTg FMD
0
30
60
90
Recognition Index (Novel/Both;sec)
Females-8.5 mos.- NOR
n.s.
D E
Wildtype
3xTg Control
3xTg FMD
0
20
40
60
80
SAB%
Females-8.5 mos.-Y- maze
n.s.
G
Wildtype
3xTg Control
3xTg FMD
0
20
40
60
80
SAB%
Males- 8.5 mos.-Y- Maze
n.s.
28
Short-term cycles of FMD regulate Nox2 cortex levels in 3xTg mice.
We also assessed whether the expression of Nox2, a central enzyme responsible for O2
-
generation
levels was affected at different timepoints of FMD administration in 8.5-month-old
male and female 3xTg mice. After 1 cycle of a 4-day FMD to young female 3xTg mice, aged
~8.5 months old we observed a significant reduction in Nox2 levels as compared to an age and
sex-matched control (p<0.05; Figure 14A). We did not observe a significant difference between
the FMD and Control groups after 4 cycles of FMD and 2 days of re-feeding in male 3xTg mice
(p=0.1497; Figure 14B). In female 3xTg mice, Nox2 levels were significantly reduced in the
3xTg group that received 5 cycles of the FMD and no refeeding as compared to both female
wildtype mice to and the 3xTg Control diet group (p<0.01, Figure 14C). In the male cohort, there
was a significant reduction in Nox2 levels in both 3xTg males on the control diet or that received
5 FMD cycles and no refeeding compared to wildtype male mice on a standard diet (p<0.01,
Wildtype vs. 3xTg FMD; p<0.05, Wildtype vs. 3xTg Control; Figure 14D).
29
Figure 14. Short-term cycles of FMD regulate Nox2 cortex levels in 3xTg mice.
(A) Quantification of Nox2 (ng/ml) in cortex extract of 8.5-month-old female 3xTg Controls (n=4) and 3xTg females after one,
4-day cycle of FMD with no re-feeding (n=10).
(B) Quantification of Nox2 (ng/ml) in cortex extract of 8.5-month-old male 3xTg Controls (n=9) and 3xTg males after 4 cycles
of FMD plus 2 days of refeeding (n=9).
(C) Quantification of Nox2 (ng/ml) in cortex extract of 8.5-month-old female C57B/6 wildtype (n=5) and 8.5-month-old 3xTg
female Control (n=6) and FMD after 5 cycles of FMD and no refeeding after last cycle (n=4) groups.
(D) Quantification of Nox2 (ng/ml) in cortex extract of 8.5-month-old male C57B/6 wildtype (n=8) and 8.5-month-old 3xTg
male Control (n=10) and FMD after 5 cycles of FMD and no refeeding after last cycle (n=4) groups.
Data are presented as mean ± SEM.
For Figure 14A and 14 B: ∗p < 0.05, Unpaired 2-tailed student’s t-test.
For Figure 14C and 14D: *p < 0.05, **p < 0.01, ***p< 0.001 compared with WT; one-way ANOVA followed by Tukey’s multiple
comparisons test.
NOX2 deletion or inhibition improves cognitive behavior, mitigates pathology progression
and reduces microglia activation.
Based on the results of the effects of FMD in mediating Nox2 levels in the 3xTg and
E4FAD mice (Figure 5A-B, 14A-D), we hypothesized that the knock-out of NADPH oxidase
could protect against cognitive decay and neuropathology in the 3xTg mouse model by reducing
O2
-
and
ONOO
-
production without preventing the protective effects of microglia.
0
2
4
6
8
10
Cortex Nox2 Levels (ng/ml)
1 cycle FMD
3xTg Female
Control
3xTg Female
FMD
*
C
Wildtype
3xTg Control
3xTg FMD
0
3
6
9
12
Cortex Nox2 Levels (ng/ml)
*
**
5 cycles FMD - Males
Wildtype
3xTg Control
3xTg FMD
0
1
2
3
4
5
Cortex Nox2 Levels (ng/ml)
**
**
5 cycles FMD - Females
D
B A
0
2
4
6
8
10
4 cycles FMD, 2 days RF
Cortex Nox2 Levels (ng/ml)
3xTg Male
Control
3xTg Male
FMD
p=0.1497
30
Edoardo Parrella (a former post doc in the Longo lab) generated 3xTg/Nox2-KO mice by
crossing Nox2-KO (Cybb-/-) mice with 3xTg mice (Figure 15A). Similar to 18.5-month-old
male 3xTg mice treated with FMD cycles (Figure 9D), 13.5-14-month-old 3xTg/Nox2-KO male
mice displayed a partial reversal of the increase in microglia density in the combined subiculum
and CA1 regions compared to 3xTg mice. Activated microglia cells as detected by CD11b
(Lynch 2009) were reduced in 3xTg/Nox2-KO mice, compared to 3xTg mice (#p<0.05; Figure
15B, bottom left).Both groups had significantly elevated levels of microglia in the combined
subiculum and CA1 regions compared to the wildtype controls (****p<0.0001, 3xTg and
***p<0.001, 3xTg/Nox2-KO; Figure 15B, bottom left). While classifying microglia activation
stages, based on a four-stages ranging from resting microglia (stage 1) to amoeboid cells (stage
4) (Zhang et al. 2011, Parrella et al. 2013), 3xTg mice showed a higher proportion of microglia
in the highly activated stages (3 and 4) when compared with the wildtype group (stage 1,
*p<0.05; stage 2, **p<0.01; stage 3, ****p<0.0001; stage 4: ****p<0.0001; Figure 15B, bottom
right). 3xTg/Nox2-KO mice showed a similar resting and stage 2 activation state, but a lower
high activation state when compared to 3xTg (stage 3, ##p<0.01; stage 4, ##p<0.01; Figure 15B,
bottom right).
With the progeny of male 3xTg/Nox2-KO mice (Figure 15A), short-term working
memory was assessed with the Y-maze apparatus. Mice of the following strains, C57B/6/Nox2-
KO, 3xTg, 3xTg/Nox2-KO and corresponding wildtype mice (C57B/6 and 129/B6), were
assessed in this test. There was a significant reduction in SAB scores with 3xTg male mice
compared to 12.5-month-old, age-matched wildtype mice and the 3xTg/Nox2-KO mice (p<0.01;
Figure 15C). These results indicate that Nox2 inactivation was able to delay the decline in
working memory in 3xTg/Nox2-KO mice.
31
Although Aβ accumulation was not modified by the inactivation of NADPH oxidase
(data not shown), we observed a significant reduction in AT8
+
hyperphosphorylated tau in the
3xTg/Nox2-KO mice as compared to 3xTg male controls (p<0.05; Figure 15D), indicating that
reduced NADPH oxidase activity and O2
-/
ONOO
-
generation represents only part of the effects
of FMD/re-feeding cycles.
Because of the changes in Nox2 expression observed in our animal models after FMD
cycles, and because of previous studies by our group and others implicating peroxynitrite or
Nox2 in mediating neurotoxicity (Xie et al. 2002, Park et al. 2008, Qin et al. 2002, Qin et al.
2006), the effects of treatment with the molecule, apocynin, on 8-month-old 3xTg mice, that
were already affected by cognitive deficits was assessed(Figure 15E). Apocynin is extensively
used as an inhibitor of NADPH-oxidase activity and of the concomitant production of ROS,
including peroxynitrite, both in vitro and in vivo. In vivo treatment for periods of 6 months were
reported(Simonyi et al. 2012, t Hart, Copray and Philippens 2014, Stefanska and Pawliczak
2008). The effect of apocynin treatment on 3xTg cognitive dysfunction was assessed using the
Y-maze apparatus and the NOR assay. When we tested these mice at ~12 months of age on the
Y-maze, 3xTg mice exhibited a significant working memory deficit in comparison with wildtype
mice, whereas SAB performance in 3xTg mice treated with apocynin was similar to that of
control mice, in agreement with our results with 3xTg/Nox2-KO mice (p <0.01, 3xTg vehicle vs
wildtype vehicle; Figure 15F). Similarly, for NOR, 3xTg mice had a significantly lower RI score
as compared to the wildtype group, whereas 3xTg apocynin-treated mice did not (p <0.05, 3xTg
vehicle vs wildtype vehicle; Figure 15G), which is also in agreement with the results obtained
with 3xTg/Nox2-KO mice.
32
Mouse treatment with apocynin did not modulate Aβ accumulation and did not affect tau
hyperphosphorylation in the hippocampus of 3xTg mice (date not shown). Apocynin treatment
had minor effects in dampening the increase in amoeboid and phagocytic states observed in the
hippocampus of 3xTg mice (stage 4, ***p<0.001 3xTg vehicle vs wildtype vehicle, **p<0.01
3xTg Apocynin vs wildtype vehicle, Figure 15H).
These results indicate that NADPH oxidase activity and probably O2
-/
ONOO
-
generation
contribute to cognitive decline, but not to Aβ accumulation nor tau hyperphosphorylation in
Alzheimer’s mouse models.
33
C
H G
E F
D
B A
34
Figure 15. NOX2 deletion or inhibition improves cognitive behavior, mitigates pathology
progression and reduces microglia activation.
(A) 3xTg/Nox2-KO mice generation and experimental design. The experimental design of the tests conducted on 3xTg/Nox2-KO
and control mice is depicted, as well as a schematic representation of the breeding strategy used to develop 3xTg/Nox2-KO mice
and corresponding wildtype (mixed background 129/B6/B6). The mice were euthanized for pathology at 13-14 months.
(B) Representative images showing CD11b-ir microglia in hippocampus sections of 13-14-month-old male wildtype (129/B6
background), 3xTg and 3xTg/Nox2-KO mice (top). Quantification of density of CD11b-ir cells in hippocampus CA1 and
subiculum combined brain regions of wildtype (129/B6 background), 3xTg and 3xTg/Nox2-KO (bottom left; n=4-8
animals/group). Percentage of different microglia activation stages (from 1 to 4) of wildtype (129/B6 background), 3xTg and
3xTg/Nox2-KO mice. (bottom right; n=4-8 animals/group).
(C) Wildtype (129/B6 background), 3xTg and 3xTg/Nox2-KO male mice were tested with the Y-maze apparatus (12.5 months of
age). SAB scores obtained through Y-maze task are shown (n=7-23/group).
(D) Representative images showing AT8 antibody (recognizes abnormally phosphorylated tau) immunoreactivity in hippocampus
of 13-14 months old male 3xTg and 3xTg/Nox2-KO mice (left). Quantification of total AT8-immunoreactive cells in
hippocampus of 13-14-month-old male 3xTg and 3xTg/Nox2-KO mice (n=10-13/group) (right).
(E) Experimental design of apocynin treatment. 8-month-old wildtype (129/B6 background) and 3xTg mice were treated with
apocynin-dissolved drinkable water or apocynin-free water for 6 months. During the final 4 weeks of treatment the animals were
tested using the Y-maze apparatus, and NOR and Rotarod assays. After completion of the behavioural tasks, the mice were
euthanized, and their brains analysed.
(F) Comparison of SAB scores between wildtype (129/B6 background) vehicle, 3xTg vehicle and 3xTg apocynin- treated mice
using the Y-maze apparatus. (n=10-13 animals/group).
(G) Comparison of RI values between wildtype (129/B6 background) vehicle, 3xTg vehicle and 3xTg apocynin- treated mice
during NOR test. (n=10-13 animals/group).
(H) Percentage of different microglia activation stages (from 1 to 4) of wildtype (129/B6 background) vehicle, 3xTg vehicle and
3xTg apocynin-treated mice. (n=5-10 animals/group).
For Figure 15B and 15H: *p < 0.05, **p < 0.01, ***p< 0.001 compared with wildtype; #p < 0.05, ##p < 0.01 for 3xTg/Nox2-KO
vs 3xTg, one-way ANOVA followed by Tukey’s multiple comparisons test (15B, bottom left) and Fisher’s least significant
difference test (15B, bottom right and 15H).
For Figure 15D: ∗p < 0.05, Unpaired 2-tailed student’s t-test.
For Figure 15C, 15F and 15G: ∗p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Fisher’s least significant
difference test.
Images were taken at 20x magnification.
Short-term cycles of FMD increase oligomeric Aβ42 internalization by IBA-1 positive
microglia isolated from 8.5-month-old 3xTg mice.
In order to investigate the primary cause of the observed effects of FMD cycles on
amyloid pathology, we isolated microglia from primary mixed glia cultures from the whole
brains (except the cerebellum) of 8.5-month-old male and female 3xTg mice after they were
administered 5 cycles of FMD or control diet and added oligomeric Aβ42 to the microglia to
assess whether FMD can enhance the uptake of oligomeric Aβ42 by IBA-1 positive microglia
compared to the control diet. We found that the oligomeric Aβ42 localizes to the cytosol of the
microglial cells (Figure 16A and 16C). IBA-1 positive microglia isolated from the brain of 8.5-
month-old 3xTg male mice after FMD cycles starting at 6.5 months of age internalized
35
significantly more Aβ42 compared to the IBA-1 positive microglia from the control group
(p<0.05, Figure 16B). There was no significant difference in the internalization of Aβ42 in the
IBA-1 positive microglia from the brain of 8.5-month-old 3xTg female controls versus FMD
(Figure 16D).
A possible link between FMD and inhibition of Aβ accumulation could be via the
increase in oligomeric Aβ42internalization by IBA-1 positive microglia.
Figure 16. Short-term cycles of FMD increase oligomeric Aβ42 internalization by IBA-1
positive microglia isolated from 8.5-month-old 3xTg mice.
(A) Representative images of IBA-1 positive microglia from mixed glia cultured from the whole brains of 8.5-month-old male
3xTg Control and FMD mice that have taken up fluorescently labeled oligomeric Aβ 42.
(B) Quantification of Aβ 42 internalization in IBA-1 positive cells (% area) isolated from 8.5-month-old male 3xTg Control and
FMD mice (n=1/group; 9 images/well, 36 images per treatment group)
B
C D
A
3xTg Control 3xTg FMD
0
50
100
150
200
Internalized Amyloid beta 42 / IBA+ (% Area) Males- 5 cycles FMD, 4 days post RF
*
3xTg Control 3xTg FMD
0
50
100
150
200
250
Internalized Amyloid beta 42 / IBA+ (% Area)
Females- 5 cycles FMD, 4 days post RF
p=0.1093
Hoechst
HiLyteᵀᴹ Fluor 555-labeled
Amyloidβ 42 (Aβ42)
IBA-1
FMD Female
Merge
Control Female
Hoechst
HiLyteᵀᴹ Fluor 555-labeled
Amyloidβ 42 (Aβ42)
IBA-1
FMD Male
IBA-1
Merge
Control Male
36
(C) Representative images of IBA-1 positive microglia from mixed glia cultured from the whole brains of 8.5-month-old 3xTg
Control and FMD female mice that have taken up fluorescently labeled oligomeric Aβ 42.
(D) Quantification of Aβ 42internalization in IBA-1 positive cells (% area) isolated from 8.5-month-old male 3xTg Control (n=1)
and FMD (n=2) mice (9 images/well, 27-45 images/treatment group).
For Figure 16B and 16D: ∗p < 0.05, Unpaired 2-tailed student’s t-test.
Data are presented as mean ± SEM
Images were taken at 40x magnification.
37
2.3 Discussion
Results obtained from two AD transgenic mouse models (E4FAD and 3xTg) indicate that
FMD cycles reduce the levels of Aβ and hyperphosphorylated tau, microglia number and markers
of neuroinflammation to attenuate cognitive decline. Supporting our FMD study, the results
obtained with the 3xTg/Nox2-KO mice and apocynin treated 3xTg mice, indicate that the positive
effects of FMD are through the reduction of the density of the microglia and the modulation of
their activity. Thus, FMD allows the microglia to perform protective functions such as the
scavenging of Aβ along with reducing the production of toxic Nitrogen Oxygen species such as
Superoxide and Peroxynitrite (O2
-
/ONOO
-
).
To address the mechanistic causes of the effects of FMD on Aβ pathology, we investigated
the microglia–dependent uptake of oligomeric Aβ42 in microglia obtained from FMD versus
control mice. We show that in males, there is a significant improvement in Aβ uptake by
microglia isolated form FMD-treated mice compared to control mice. In females, we only
observed a trend for this effect (Figure 16D). A recent study using 18-kDa translocator protein
positron-emission-tomography (TSPO-PET) showed that microglia activation is higher in App
NL-
G-F
female mice compared to male mice in response to amyloidosis (Biechele et al. 2020). There
is also a higher proportion of activated response microglia (ARMs) cells in 6-month-old and
older App
NL-G-F
female mice compared to male mice. This indicates that the female microglia in
an amyloid model of AD are activated earlier than those in male mice (Sala Frigerio et al. 2019)
and could explain the differences in the effects of FMD on the uptake of oligomeric Aβ42 in
male versus female microglia isolated from 3xTg mice. Based on current studies as well as on
our past studies with essential amino acid restriction, we believe that although FMD cycles can
38
affect Aβ levels, their major effect is related to altering Aβ-dependent effects, such as those on
hyperphosphorylated tau as well as neuroinflammation and not Aβ levels.
Previous research suggests that the NADPH oxidase complex has an important and a specific
role in modulating microglial activation, and that reactive oxygen species production by NADPH
oxidase as well other sources is involved in Aβ-dependent microglia proliferation by releasing
pro-inflammatory cytokines (Jekabsone et al. 2006). In our study of the brains of 3xTg mice, FMD
cycles partially reversed the strong effect of the AD mutations on increasing microglia numbers
and also reduced Nox2 levels. Along with our previous results showing that both O2
-
and ONOO
-
promote Aβ- and microglial-dependent neurotoxicity (Longo et al. 2000, Xie et al. 2002), the
results obtained in this study indicate that some of the protective effects imparted by the FMD
cycles are associated with reduced microglial and Nox2 activation/levels. Therefore, a reduction
in ROS (O2
-
and ONOO
-
)
generation and toxicity by FMD cycles, may also lead to an improvement
in cognition, decrease tau phosphorylation and affect synaptic plasticity (Schematic 4).
Previous research in the Longo lab has shown that FMD cycles alternating with a re-feeding
period can promote stem cell-dependent regeneration in different systems of the body including
the nervous systems (Brandhorst et al. 2015, Choi et al. 2016, Rangan et al. 2019, Cheng et al.
2014). We show that FMD cycles increase the expression and generation of proliferating neural
stem cells (as indicated by an increase in Ki67
+
Sox2
+
expression). However, the contribution of
neural stem cells to the attenuation of cognitive decline or Nox2 remains to be investigated.
To summarize, we propose a role for periodic FMD cycles in a reducing AD associated
pathology, neuroinflammation and Nox2 allowing for the microglia to scavenge Aβ consistent
with studies in a different AD mouse model (Bruce-Keller et al. 2011). Our collaborators in
Genoa, Italy have an ongoing, placebo-controlled, randomized clinical trial in patients with MCI
39
or early-stage AD that provides initial evidence indicating that FMD cycles were feasible and
safe in the twelve patients tested thus far. The enrollment of all patients in this trial along with
data on cognition in this clinical trial study will help to conclude whether FMD cycles are their
efficacious against cognitive decline and disease progression in AD patients.
40
2.4 Materials and Methods
Mouse models
All animal protocols used in this study were approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of Southern California (USC). Mice were maintained in a
pathogen-free environment, group-housed in cages of up to 5 animals/cage regulated with
constant temperature, humidity and 12-hour light/dark cycles, along with unlimited access to
water. Mice that appeared weak and/or showed signs of illness were excluded from the
experiments. Body weight and food intake of individual mice was measured every day during the
FMD and at least once during the refeeding period.
3xTg Mice (long-term study)
3xTg breeding pairs were obtained from Frank LaFerla, PhD (UC Irvine) under an MTA
agreement. For the long-term study using 3xTg mice, male and female 3xTg mice were divided
into 3 groups - the ad libitum-fed control (Control) group, the FMD group, or a 7-day protein-
restricted diet (approximately 4% protein in composition relative to carbohydrates and fat) group
(4% PR). They were administered the diets starting at 3.5 months of age till ~ 18.5 months of
age.
3xTg Mice (short-term studies) - For the short-term study using 3xTg mice, male 3xTg mice
were divided into 2 groups- the ad libitum-fed control (Control) group or the FMD group. They
were given the diet starting at ~6.5 months of age till ~8.5 months of age. Each FMD cycle
consisted of 4 days of the diet followed by 10 days of refeeding. 8.5-month-old male 3xTg mice
were sacrificed 2 days after refeeding, after the 4
th
FMD cycle. 8.5-month-old female 3xTg mice
were divided into the Control or the FMD group to assess the effect of 1 cycle of FMD (4 days)
41
and were euthanized prior to refeeding. Wildtype mice (C57BL/6) were purchased from Jackson
Laboratory (Bar Harbor, ME) at 8.5 months of age. Another short-term study was carried out in
which male and female 3xTg mice were given FMD starting at 6.5 to ~8.5 months for a total of 5
cycles and the mice were euthanized after the 5
th
cycle before refeeding.
E4FAD Mice - E4FAD breeding trios were obtained from Mary Jo LaDu, PhD (University of
Illinois) under an MTA agreement. Starting at 3 months of age, female E4FAD were divided into
the ad libitum-fed control group or the FMD group until ~ 7-7.5 months of age.
3xTg/Nox2-KO Mice- The following male mice were used in this study: 3xTg, Nox2
_
KO,
3xTg/Nox2-KO
and wildtype C57BL/6/129S (129/B6).
Nox2-KO mice present a null allele of the X-linked Cybb gene which encodes the Nox2 subunit
of the NADPH oxidase complex which result in a lack of phagocyte superoxide production
(Pollock et al. 1995). Nox2-KO and WT (C57BL/6) mice were purchased from Jackson Laboratory
(Bar Harbor, ME). Colonies of 3xTg mice and related wildtype (C57BL/6/129S) were established
and bred at University of Southern California. 3xTg/Nox2-KO mice were generated for this study
by Edoardo Parrella as described below:
3xTg male mice were crossed with female KO mice for Cybb, the gene coding for the Nox2
subunit of NADPH oxidase. Since the Cybb is an X-linked gene, all the male progenies were
hemizygous for the genes related to Alzheimer’s disease and lacked the Nox2 subunit of
NADPH oxidase and the F1 male offspring were back-crossed to homozygosity for APP/Tau and
PS-1 genes. The correct genotype of the mice was determined by PCR(Pollock et al. 1995, Guo
et al. 1999) or qPCR by comparison of the ΔCt values of each unknown sample against known
homozygous as well as hemizygous controls. In order to obtain the 3xTg/Nox2-KO background,
42
C57BL/6/129S males that had the 129/B6, 3xTg background were crossed with C57BL/6 mice
with a B6, Nox2-KO background. In order to equalize the genetic background, the F1 offspring
were backcrossed for four generations.
Mouse Diets
The control diet used in this study was PicoLab Rodent Diet 20 (LabDiet). The FMD
administered to the mice is based on a screening of nutrients that identified certain ingredients
that allow for nourishment during periods when the calorie consumption is low (Brandhorst et al.
2015). The FMD formulation given to the mice in the AD study consisted of two different
components namely, the day 1 diet and day 2–4/5 diet. In the long-term study, FMD was fed to
male 3xTg mice for 4 days while female 3xTg mice were on the diet for 5 days whereas in the
short term 3xTg study, both male and female 3xTg mice were given 4 days of the diet. The day 1
diet consisted of a mix of various low-calorie broth powders, a vegetable medley powder, extra
virgin olive oil, and essential fatty acids. Day 2–4/5 diet consisted of the same ingredients as in
day 1, along with the addition of glycerol. The diet formulations of both Day 1 and Day2-4/5
contained hydrogel (Clear H2O). The macronutrient composition of Day 1 is approximately 55%
fat, 36% carbohydrate and 9% protein and Day 2-4/5 is composed of approximately 43.3% fat,
47.4% carbohydrate, and 9.3% protein. On Day 1, the mice consumed any amount between
12.36 kJ-17.31 kJ while on Day 2-4/5, the mice consumed any amount between 8.17kJ – 11.44
kJ a day.
The 7-day 4% protein-restricted diet (4% PR) based on previous amino acid restriction studies
with 3xTg mice (Parrella et al., 2013) was composed of approximately 75.67% carbohydrate,
43
3.90% casein protein, and 20.43% fat. The 3xTg male and female mice in the 4% PR group
consumed any amount between 19.36 kJ – 27.10 kJ per day.
Mice in the study consumed all the food that was given on every day of the FMD and did not
show any signs of food aversion. After day 4 or 5 of the FMD or day 7 of the 4% Protein
Restriction cycle, the mice were given PicoLab Rodent Diet 20 chow ad libitum for either 10, 9,
or 7 days prior to starting another diet cycle. Before administering the FMD or 4 %PR, mice
were transferred into fresh cages in order to avoid feeding on residual chow and coprophagy.
Apocynin Treatment
Male 3xTg and wildtype mice with a 129/B6 background were group housed (3-5 per cage) and
were treated with 1 mg/ml apocynin (Sigma-Aldrich) that was dissolved in drinking water or
apocynin-free water for 6 months, starting at 8 months of age. The mice in the study were
assigned to the experimental groups by randomization. Dose and route of administration of
Apocynin were selected according to previous reports (Harraz et al. 2008). The time point of
Apocynin treatment was chosen based on previous research showing that 8-month-old 3xTg
mice have impaired memory and Aβ and tau pathology develops in their brains (Mastrangelo and
Bowers 2008, Oddo et al. 2003). In order to prepare the drug treatment for the mice, Apocynin
powder was first dissolved into hot (~60°C) sterile water and then allowed to cool to room
temperature before being given to the mice. Taking into consideration that the average daily
water intake approximately 4 ml/mouse (Bachmanov et al. 2002), the mg/kg dose of Apocynin
that was given to the mice was estimated approximately 150 mg/kg body weight/day. Apocynin
was replaced every week since between 5-7 days any decay of the drug is detectable (Harraz et
al. 2008).
44
The mice in the study were weighed weekly and monitored for abnormal changes during the
treatment period so that any possible adverse effects due to the Apocynin treatment could be
detected. During the final three weeks of treatment (around 14 months of age), while they were
still under treatment with Apocynin, the mice were tested in the Y maze and the NOR behavioral
tests.
Behavior Tests
SAB using the Y‐maze- Short‐term working memory was examined by spontaneous alternation
behavior in the Y‐maze (Parrella et al. 2013). The Y‐maze was constructed from black plexi-
glass and was designed with three identical arms (50 × 9 × 10 cm), originating from a central
triangle (8 cm on each side) and spaced 120° apart from each other. Mice were placed at the
bottom of one of the arms of the maze at the start of the test and then were allowed to freely
explore the maze for 8 minutes. The total number of arm entries and arm choices (defined as
forepaws fully entering the arm) were recorded. SAB was calculated as the proportion of
alternations (defined as an arm choice different from the previous two choices), divided by the
total number of alternations.
Novel Object Recognition (3xTg mice Study)- The novel object recognition (NOR) test is a
test of short-term memory and is used to assess the ability of mice to recognize a novel object in
a familiar environment (Parrella et al. 2013). NOR includes a 5-minute habituation phase on day
one and two trial phases for 5 minutes each on day 2 (5 minutes apart) for each mouse. On Day
one, each mouse was placed into a rectangular cage (50 x 50 x 40 cm) made of black plexiglass
for 5 minutes without any objects in order to become familiar with the environment. All the mice
tested in the study were always placed in the NOR apparatus facing the wall at the middle of the
45
front segment. Time taken to explore the objects was counted when the mouse was in close
contact with an object by either whisking, sniffing, rearing on or touching the object along with
pointing its nose toward the object at a distance of less than 2 cm. Sitting or standing on top of
the object was not counted toward the time taken to explore the object. In trial 1 of day 2, mice
explored the area of the NOR apparatus with two identical objects for 5 minutes. Mice were then
placed back in their home cage and in order to control for odor cues, the arena and the objects
were thoroughly cleaned with 70% ethanol, dried, and ventilated for a few minutes between
mice/between trials. After an interval of 5-minutes (Sik et al. 2003), mice were placed back in
the apparatus for the second trial, where one of the objects from the first trial was replaced with a
new one. Recognition index (RI) was calculated as time the mice spent exploring the novel
object to the total time the mice spent exploring both the novel and the old objects (Parrella et al.
2013).
Novel Object Recognition (Apocynin Study)- The mice treated with apocynin or apocynin-free
water were tested for short-term spatial memory using a maze that consisted of an opaque plastic
box with the dimensions of 61 cm (length) x 36 cm (width) x 30 cm (height). On day one, the
mice were placed in the box and explored the field for 5 minutes. On Day 2, habituated mice
were placed again into the box in the presence of two identical, non-toxic objects. The mice were
allowed to freely explore the objects for 5 minutes (trial 1). The time spent exploring the objects
by a mouse was counted when there was as any physical contact with an object and/or obvious
orientation to it within 5 cm. The mice were then returned to their home cage and after 3 minutes
the mice were returned to the box where one of the familiar/identical objects was replaced by a
novel object. The mice were allowed to explore the arena for 5 minutes (trial 2) and the time
taken to explore the objects was recorded again. Recognition index (RI) was calculated as the
46
time the mice spent exploring the novel object to the total time spent exploring both the objects
(Parrella et al. 2013).
Barnes Maze- The Barnes maze protocol used here was based on previous protocols(Barnes
1988, Michán et al. 2010). The Barnes maze consisted of a platform with 20 holes (San Diego
Instruments) with 20 boxes underneath each hole. A nestlet was placed in one box (escape box,
“EB”), and this box was big enough for the mouse to enter. All the walls around the Barnes maze
had different reference cues to help the mouse learn the position of the escape box. A unique
position for the EB based on the position of one of the reference cues was randomly assigned to
each mouse and this position was the same for a specific mouse throughout the study. Mice were
acclimated to the behavior room for an hour before the test began on each day of the Barnes
maze study. All the mice in the study were trained once daily on days 0 to 7 to freely explore the
maze until either entering the EB or after the trial time of 2 minutes elapsed. On Day 0, the mice
were either classified as active or inactive. This was based on whether the mice moved around
the center of the maze or explored the holes at the periphery of the maze. During training, if the
mouse did not enter the EB by itself after the 2 minutes elapsed, it was gently guided to and
allowed to stay in the EB for 30 seconds. Throughout the duration of the study, the buzzer with a
noise level of 80 dB was always switched on when the mice were in the behavior room except
when the mouse was guided to the escape box or found it by itself. After the training, mice were
tested twice daily for 7 days and if the mouse did not find the EB after 2 minutes, it was directly
returned to its cage.
Success rate, latency, number of errors, deviation (how many holes away from the EB was the
first error), and strategies used to locate the EB were recorded and averaged from the two test
47
trials to obtain daily values for each mouse. Search strategies were classified as random, serial or
spatial and retention was assessed by testing the mice once on day 14.
Immunohistochemistry (IHC)
At the end of the studies, mice were euthanized with isoflurane, punctured in the heart for
serum, followed by intracardial perfusion with 4% paraformaldehyde (PFA). The brains were
removed immediately and post-fixed in 4% PFA for 24 hours for the long-term 3xTg mice study
and then stored in PBS containing 0.05% sodium azide. For E4FAD as well as the short-term
3xTg studies, mice were perfused with saline instead of 4 % PFA and the brains were removed
immediately and cut in half. One half of the brain was post-fixed in 4% PFA for 48 hours and
then stored in PBS containing 0.05% sodium azide. The other half of the brain was dissected for
the cortex and hippocampus, both of which were flash-frozen at -80C until further processing.
Brains from 3xTg/Nox2 mice were collected, immersion-fixed in fresh 4% paraformaldehyde/0.1
M PBS for 48 hours. They were then stored at 4 °C in PBS containing 0.2% sodium azide until
further processing. For Immunohistochemistry (IHC), hemi-brains were cut in a sagittal direction
(40 μm) using a vibratome VT1000S (Leica) and stored in PBS containing 0.05% sodium azide
solution. Every 6th section of the brain containing the hippocampus was immunostained in the
subsequent staining protocols.
3,3’-diaminobenzidine (DAB) Staining- For 3,3’-diaminobenzidine (DAB)-based protocols,
sections were first washed in TBS, pre-treated for antigen retrieval when necessary, then
incubated in methanol and H2O2 , prior to blocking in 2% BSA for 1 hour followed by incubating
with primary antibodies overnight at 4 °C. On day 2 of staining, the sections were washed in
TBS, incubated with secondary antibodies, ABC Vector Elite (Vector Laboratories), and DAB
48
kits (Vector Laboratories) before being mounted on slides and air-dried. Primary antibodies used
for 3,3’-diaminobenzidine (DAB)-based protocol were beta amyloid (1:300; Thermo Fisher),
Phospho-Tau (Ser202, Thr205) Antibody (AT8) (1:1000;Thermo Fisher), CD11b (1:500;
BioRad), Iba1 (1:500; Wako) and CD68 (1:500; Bio-Rad) . Secondary antibodies used were
biotinylated goat anti-rabbit IgG (for beta amyloid, 1:500; Vector Laboratories), biotinylated
horse anti-mouse IgG Antibody, rat adsorbed (for AT8 and Iba1, 1:500; Vector Laboratories),
donkey anti rat IgG (H+L), biotin (for CD11b, 1:250; Thermo Fisher Scientific) and biotin-SP-
AffiniPure donkey anti rat IgG (H+L) (for CD68, 1:500; Jackson ImmunoResearch).
Aβ - In order to enhance the immunoreactivity (IR) of Aβ, sections were rinsed for 5 min in 99%
formic acid before the staining protocol was carried out as described above. Imaging of sections
for Aβ was carried out as follows- selected fields of nonoverlapping immunolabeled sections of
the hippocampus (one-two fields for subiculum and two-three for CA1– Cornu Ammonis area 1)
were captured at 20X and digitized using a video capture system coupled to a microscope
(Olympus BX50 microscope and Olympus DP73 camera). Images obtained were converted into
binary/negative data using ImageJ software (NIH) software. The positive pixels equivalent to IR
area were quantified (Carroll et al. 2010).
Tau- AT8-immunoreactive neurons were defined as cells showing strong AT8 immunolabeling
over most of the cell surface and were counted within the subiculum and CA1 regions of the
hippocampus. Images of sections for Tau were captured with a video capture system coupled to a
microscope (Olympus BX50 microscope and Olympus DP73 camera) at 20X.
49
CD11b, Iba1 and CD68 (Microglia Staining)- CD11b-immunoreactive (ir), Iba1+ and CD68+
microglia cells were defined as cells covered by CD11b, Iba1 or CD68 immunostaining over the
cell body and processes. Sections were either pre-treated with 10mM EDTA (pH 6) or Citric
Acid Buffer (pH 6) at 95ºC for 10min before proceeding with DAB Immunohistochemistry.
CD11b-ir, Iba1+ and CD68+ microglia were counted in the subiculum and CA1 regions of the
hippocampus. There was no pre-treatment with 10mM EDTA (pH 6) or Citric Acid Buffer (pH
6) for required the CD68 Antibody.
Four stages of microglia activation based on criteria established in previous studies was used to
classify the microglia with different morphological characteristics (Zhang et al. 2011, Kreutzberg
1996, Crews and Vetreno 2016):
•
Stag
e 1: Resting microglia. •
Stag
e 2: Activated ramified microglia.
•
Stag
e 3: Amoeboid microglia • Stage 4: Phagocytic cells.
To determine the percentage of CD11b-ir or Iba1+ cell number, microglia in the different
activation stages were counted and divided by the total CD11b-ir or Iba1+ cell number.
Images for CD11b-ir, Iba1+ and CD68+ microglia were captured at 20X, with a video capture
system coupled to a microscope (Olympus BX50 microscope and Olympus DP73 camera).
50
Immunofluorescent Staining- Sections were rinsed in PBS for 5 minutes, denatured in 2N HCl
at 37°C for 20 minutes followed by neutralization with 0.1M boric acid for 10 minutes (for Ki67
antibody) and blocking with 2% Normal Donkey Serum (NDS; Jackson ImmunoResearch) for 1
hour at room temperature. Sections were incubated with primary antibodies overnight at 4°C
along with 2% NDS and 0.3% triton. On day 2, sections were rinsed in PBS for 10 min, followed
by incubation with secondary antibodies. Nuclei were stained with Hoechst 33342 (Thermo
Fisher), washed in PBS, and mounted on slides (except for GFAP+ Sox2+ staining as the
mounting medium contained DAPI). Slides were cover-slipped with anti-fading polyvinyl
alcohol mounting medium with DABCO (Sigma-Aldrich) or VECTASHIELD® HardSet™
Antifade Mounting Medium with DAPI (Vector Laboratories) immediately after the sections
were mounted on the slides. Primary antibodies used in this immunofluorescent staining protocol
were Ki67 (1:200; Thermo Fisher), Sox2 (1:200; Abcam) and GFAP (1:200; Cell Signaling).
Secondary antibodies used were donkey anti-rat-488 (Ki67 and GFAP, 1:400; Thermo Fisher)
and donkey anti-rabbit-594 (for Sox2, 1:400; Thermo Fisher). Co-expression of Ki67+Sox2+ and
Sox2+ GFAP+ was confirmed by fluorescent microscopy and stereological counting methods
were used to count the number of co-localized cells as well the number of Sox2+, Ki67+ or
GFAP+ cells.
Neurogenesis Markers- The levels of Sox2, Ki67 and GFAP were quantified in the dentate
gyrus while the images were captured at 20X and captured with a video capture system coupled
to a microscope (Olympus BX50 microscope and Olympus DP73 camera) or with the BZ-X710
All-in-One Fluorescence Microscope (Keyence). The images obtained were analyzed with
ImageJ (NIH). Individual Sox2
+
, Ki67
+
and GFAP
+
cells, as well as Ki67
+
Sox2
+
and
GFAP
+
Sox2
+
co-stained cells, were counted if they were located within the subgranular zone
51
(SGZ) and up to the inner third of the granule cell layer. Every sixth section of the mouse hemi-
brain containing the hippocampus was stained. The numbers of cells quantified are the averages
taken from the total number of sections stained for each hemi-brain.
Aβ Protein Extraction
A serial extraction process previously described was used to produce TBS-soluble and TBS plus
triton-soluble fractions of Aβ peptides (Aβ38/40/42) (Youmans et al. 2011) that were extracted
from the cortex of hemi-brains of 7-7.5 months old female E4FAD mice.
Immunoprecipitation
Magnetic Dynabeads (Invitrogen) were re-suspended and 50 μl was transferred to Eppendorf
tubes. The magnetic beads were separated from supernatant using a magnet and the supernatant
was removed. The Antibofy used was the Rat anti- mouse CD11b antibody (1 μg/μl , Biorad)
that was diluted in 200 μl of Ab Binding and Washing Buffer. The immunoprecipitation
procedure was carried out using the Dynabeads™ Protein G Immunoprecipitation Kit (Thermo
Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. 500 ug of target
antigen (Aβ42) was added to the magnetic bead antibody complex. Samples were then
mixed with 10 µL of pre-mixed NuPAGE™ LDS Sample Buffer and NuPAGE Sample
Reducing Agent (Invitrogen) to ensure the resuspension of the magnetic bead-Ab-Ag complex
after the elution with the elution buffer and heated for 10 min at 70ºC. The supernatant obtained
was transferred to a clean tube and prepared for western blot as described in the Western Blot
section.
52
Western Blot
The hemi-cortex from E4FAD mouse brains was homogenized in Eppendorf tubes (kept on ice)
using the homogenization solution containing 300 ul of RIPA buffer (Thermo Fischer Scientific)
with protease and phosphatase inhibitor cocktails. The homogenized samples were placed on an
orbital shaker at 4°C overnight and spun the next day at 12,000 rpm for 20 min at 4°C. The
protein concentration from each sample was estimated using BSA as working standard. In order
to ensure that the same amount of each sample assayed, equal amounts of protein (40 µg) were
heat-denaturized in NuPAGE LDS sample- loading buffer (Invitrogen) for 5 minutes at 95°C.
The samples were loaded onto the wells of an SDS-PAGE gel and then transferred to PVDF
membranes (Sigma-Aldrich, MO, USA). The blocking reagents used for the PVDF membranes
were Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk for NOX2,
Aβ42 and Tris-buffered saline (TBS) containing 0.05% Tween and 2% BSA for Vinculin.
Primary Antibodies incubated overnight were directed against NOX2 (Rabbit monoclonal,
1:5000, Abcam), Aβ42 (Rabbit monoclonal, 1:500, Abcam) or Vinculin (Mouse monoclonal,
1:1000, Millipore). The secondary antibodies were anti-rabbit or anti-mouse Peroxidase
conjugated IgG. Membrane-bound protein complexes were detected by HyGLO™
Chemiluminescent HRP Detection Reagent (Denville Scientific) and protein loading was
normalized to the expression of Vinculin. Quantification of the amount of protein was performed
by densitometric analysis using Imagelab 6.0 software.
Nox2 ELISA
53
Nox2 levels in the hemi-cortex of young 3xTg mice was measured using a Rat CYBB / NOX2 /
gp91phox ELISA Kit (LSBio), according to manufacturer’s instructions.
In vitro microglia-Aβ42 assay-
Mixed glia were cultured from the brains of 8.5 month old 3xTg male and female mice that were
given either a control diet or 5 cycles of FMD with 4 days of refeeding (n=1-2 mice/group)
starting at 6.5 months till 8.5 months of age. The brains were perfused using DPBS and the
whole brain (except the cerebellum) from each of the 5 mice (3 female mice- 2 FMD and 1
Control and 2 male mice- 1 FMD and 1 Control) were placed in 5 ml of DMEM/F12 (Gibco) and
stored on ice. The procedure for the isolation of microglia was as follows-Whole brains
suspended in DMEM/F12 were mechanically and enzymatically dissociated by first mincing
with a sterile razor blade and then incubated with 2 ml of serum free DMEM/12 media
containing 10 units/ml papain for 25 min followed by the addition of 40 ul of Dnase (10 mg/ml
stock, final 0.2 ug/ml) for 5 mins at 37°C. At the end of the incubation with papain and Dnase,
the enzymatic activity was quenched by addition of 500 μL of fetal bovine serum followed by
addition of 2 ml of serum free media at room temperature. Large clumps of tissue were broken
up by pipetting using a 1 ml pipette. After the cell pellets settle for two minutes, the supernatant
was the transferred to a fresh falcon tube and this step was repeated twice more. Isotonic Percoll
(ISP) was prepared by mixing nine parts of Percoll (Percoll PLUS, Cytiva) with one-part 10×
HBSS and was added to the cell suspension at a final concentration of 20%.
In order to collect the glial-enriched fraction, the cell suspension containing 20% ISP was
centrifuged for 20 min at 700×g. The supernatant was aspirated, and the cell pellets were re-
suspended in 8 ml of DMEM/F12 supplemented with 10% FBS and a penicillin G and
54
streptomycin cocktail. The suspension was then transferred through a 100 μm cell strainer on
petri plates coated with 10 μg/ml of Poly-D Lysine. Primary cell cultures were maintained at
37°C in a humidified incubator for 10 days with room air supplemented with 5% CO2. The
media was replaced 3 times per week in order to remove cell debris.
After the primary mixed glial cultures were incubated for 10 days, 2500 microglial cells per well
were seeded on 96 well plates coated with 10 ug/ml of Poly-D Lysine and incubated for two days
at 37°C in a humidified incubator with room air supplemented with 5% CO2.
HiLyteᵀᴹ Fluor 555-labeled Amyloidβ 42 (Aβ42) (AnaSpec, Liège, Belgium) was first dissolved
in 100% 1,1,1,3,3,3-Hexafluoro-2-propanol and then dried followed by evaporation. The peptide
film of was Aβ42 was dissolved in 10 mM NaOH and neutralized by adding pH 7.4 PBS to a final
concentration of 20 μM. After the sample was vortexed for 30s, Aβ42 was incubated at 4℃ for
24h for oligomerization and was used for assay immediately where it was added to the seeded cells
on a 96 well plate for an hour to quantify the levels of Amyloid beta uptake in microglia isolated
from the FMD compared to the control group. After the addition of oligomeric Aβ42, the
microglia were fixed with 4 % PFA and stained with IBA-1 (1:200; Wako) to confirm the cell
type. The Secondary antibody used was donkey anti-mouse-488 (for IBA-1, 1:500; Thermo
Fisher). Images are taken with the BZ-X710 All-in-One Fluorescence Microscope (Keyence) and
analyzed using ImageJ (NIH). The area of Amyloid beta 42 to IBA-1 area was quantified to
determine the percentage of Aβ 42 uptake in control versus FMD group.
Quantification and Statistical Analysis - The software used for statistical analysis was
GraphPad Prism v.8. The figure legends describe the statistical tests used, value of n for each
experimental group, and what n represents for each experiment. All data in the figures are
55
expressed as the mean ± SEM. All statistical analyses were two-sided and p-values <0.05 were
considered significant (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Statistical analysis
between 2 groups was tested with unpaired 2-tailed student’s t-test comparison, and one-way or
two-way ANOVA followed by Bonferroni, Tukey’s multiple comparison test, or Fisher’s test
was used among multiple groups. Animals were randomly assigned to experimental groups and
were excluded from the analysis only if they fell ill based on protocol or did not move (in the
Barnes maze behavior study).
56
Chapter 3: Dietary Interventions in Parkinson’s disease (PD)
Parkinson’s disease is the second most common neurogenerative disorder. It affects 2-3
percent of the population after 65 years of age and is twice as common in men than in women in
most populations. The main neuro pathological hallmarks of Parkinson’s disease are intracellular
inclusions that contain aggregates of α‐synuclein (Lewy bodies), striatal dopamine deficiency
and neuronal loss in the substantia nigra. The motor symptoms of Parkinson’s disease in patients
are the presence of bradykinesia, rigidity and resting tremor while the non-motor symptoms
include cognitive impairment, sleep disorders, depression and autonomic dysfunction. Majority
of the cases of Parkinson’s disease are sporadic while the inherited forms of the disease account
for 5-10 % of the cases but their study has provided valuable insights into the pathogenesis of the
disease (Poewe et al. 2017).
3.1 Pathogenesis of Parkinson’s disease
In heritable forms of Parkinson’s disease, there is an existence of point mutations and
multiplications of the gene encoding for alpha synuclein (SNCA). Alpha synuclein, a 140 amino
acid protein, has roles in mitochondrial function, intracellular trafficking, synaptic vesicle
dynamics (Bendor, Logan and Edwards 2013). However, in Parkinson’s disease, Alpha
Synuclein undergoes a pathogenic process in which soluble monomers form oligomers that
combine to form protofibrils and eventually large insoluble fibrils which form the aggregates that
make up Lewy bodies (Poewe et al. 2017). A decline in proteolytic pathways with age could
contribute to the accumulation of Alpha Synuclein in PD patients (Kaushik and Cuervo 2015,
Xilouri, Brekk and Stefanis 2013). The ubiquitin-proteasome system and the Lysosomal
Autophagy System (LAS) are very important in maintaining the homeostasis of alpha synuclein.
57
As part of the LAS, Macroautophagy and chaperone mediated autophagy mediate the
degradation of Alpha synuclein. Inhibition of either system can lead to the accumulation of
Alpha Synuclein (Xilouri et al. 2013, Brundin et al. 2008). Another mechanism for Alpha
synuclein aggregation is the prion hypothesis. This hypothesis posits that once alpha synuclein
aggregates are formed in a neuron, they can be transported to other brain regions and be taken up
by neighboring neurons where they seed the aggregation of Alpha synuclein in the new cell
(Angot et al. 2010, Brundin, Melki and Kopito 2010).
Mitochondrial dysfunction is also an important element in the pathogenesis of
Parkinson’s disease. Alpha Synuclein aggregation and mitochondrial dysfunction have been
reported to exacerbate each other (Poewe et al. 2017, Schapira 2007, Bose and Beal 2016). In
patients with Parkinson’s disease, the activity of mitochondrial complex 1 is reduced (Schapira
2007, Bose and Beal 2016) while the target genes of Peroxisome proliferator-activated receptor-γ
(PPARγ) co-activator 1α (PGC-1α), a mitochondrial master transcriptional regulator, are under
expressed in Parkinson disease (Zheng et al. 2010). PGC-1α downregulation leads to an increase
in alpha synuclein oligomerization in vitro while its genetic overexpression rescued against
Alpha Synuclein mediated toxicity. Eschbach et al, proposed that PGC-1α downregulation and α-
syn oligomerization form a vicious circle, in which they can influence or potentiate each other
(Eschbach et al. 2015). LRRK2 mutations that are found in patients with inherited forms of the
disease are associated with changes in autophagy as well as with mitochondrial impairments
(Bose and Beal 2016). Proteins encoded by autosomal recessive Parkinson’s genes PARK2 and
PINK1 play an important role in the clearance of damaged mitochondria through mitophagy
(Pickrell and Youle 2015).
58
Oxidative stress has been reported to result from mitochondrial dysfunction. Mutations
in DJ1(PARK7) that causes early onset autosomal recessive Parkinson’s disease, are associated
with increased cellular oxidative stress. Along with mitochondrial dysfunction, increased
oxidative stress that can result from increased levels of cytosolic dopamine and its metabolites,
can lead to the depletion of lysosomes and impair LAS. This demonstrates the connection of
several pathogenic pathways in Parkinson’s disease. Neuroinflammation is another feature of
Parkinson’s disease and is essential to the pathogenesis. Alpha synuclein aggregation induces
both innate and adaptive immunity in Parkinson’s disease. Neuroinflammation can promote the
misfolding of Alpha Synuclein (Poewe et al. 2017).
Schematic 1. Molecular mechanisms underlying Parkinson’s disease.
The figure depicts the interactions between the main molecular pathways that are involved in the pathogenesis of Parkinson’s
disease namely-α-synuclein proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport and
neuroinflammation (Poewe et al. 2017).
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3.2 Autophagy, Mitophagy and Parkinson’s disease
It has been well established that nutrient depletion, growth factor deprivation and low
energy are autophagy inducers that result in mammalian target of rapamycin complex 1
(mTORC1) inhibition and AMP-dependent protein kinase (AMPK) activation which in turn
regulates the mammalian homologs of the C. elegans uncoordinated-51 kinase (ULK1) complex
through phosphorylation. Induction of ULK1 activates VSP34 which in turn leads to
Phosphatidylinositol 3-phosphate (PI3P) synthesis in pre-autophagosomal structures. PI3P assists
the recruitment of the ATG12-ATG5-ATG16L1 complex which is necessary for the conjugation
of LC3 to phosphatidylethanolamine (PE) in membranes. Along with mTORC1 inactivation,
transcription factor EB (TFEB) translocates to the nucleus and its activation that leads to the
transcription of many autophagy and lysosomal genes. In Parkinson’s disease, mutations in
certain genes can lead to impaired autophagosome formation (Alpha Synuclein, VPS35) or
disrupted lysosomal function (ATP12A2, SYT11, GBA, Alpha Synuclein, VPS35). Autophagy
inducers are important as therapeutics for PD neurodegenerative disorders and Trehalose is one
such autophagy inducer that acts via AMPK activation (Menzies et al. 2017).
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Schematic 2. Mammalian Autophagy Pathway.
Key components of the mammalian autophagy pathway regulated by nutrient depletion, growth factor deprivation and low
energy (Menzies et al. 2017).
3.3 Current Treatments and disease modifying therapies
PD has a complex etiology and can result from a combination of genetic and
environmental risk factors. There are no treatments that are currently available to efficiently
modify the disease or slow its progression. Patients with the disease show motor as well as non-
motor symptoms. Besides Levodopa that causes adverse side effects, current treatment options
for PD include, dopamine agonists such as ropinirole or rotigotine and Monoamine Oxidase B
inhibitors such as Rasagiline and Selegiline. These treatments primarily restore dopaminergic
activity in the striatum and thus improve only the motor symptoms but not the non-motor
symptoms (Stoker, Torsney and Barker 2018, Lindholm et al. 2016). Deep brain stimulation
(DBS) is another treatment option that is very effective in controlling the movement disorder but
it also does not help with non-motor symptoms like the previously mentioned treatment options
(Stoker et al. 2018). Microglia secrete a number of molecules and pro-inflammatory cytokines
such as Interleukin-1b, interferon-c, Tumor necrosis factor Alpha and Nitric Oxide which can
exacerbate the disease. Microglia activation and pro-inflammatory cytokines are targets for
61
therapy. Neurotrophic factors such as Glial derived Neurotrophic factor has neuroprotective
potential in PD while the Glucagon-like peptide receptor (GLP-1R) is also shown to be
neuroprotective in PD models. A GLP-1R agonist (exenatide) has been given to PD patients in
clinical trials and it has shown to influence motor and cognitive functions in patients with
moderate disease (Lindholm et al. 2016). Exenatide, has been known to activate glucose-
dependent insulin secretion, slow gastric emptying, and is approved for the treatment of type 2
diabetes mellitus (Deuschl and de Bie 2019). Like the GLP-1R agonist, intermittent energy
restriction and exercise are lifestyle changes that also increase insulin sensitivity and may
enhance neuronal adaptive stress responses and increase neurotrophic signaling, DNA repair,
proteostasis and mitochondrial biogenesis (Mattson 2014).
3.4 Animal models of PD
Animal models in PD can be divided into two types- those models developed by the use
of environmental or synthetic neurotoxins or genetic models that utilize the expression of PD-
related mutations. Neurotoxic models include 1- methyl-1,2,3,6-tetrahydropyridine (MPTP), 6-
hydroxydopamine (6-OHDA), Paraquat (PQ), Rotenone or Reserpine. A main feature of the
neurotoxin models is the ability to produce oxidative stress and cause cell death in dopaminergic
neurons. The 6-OHDA mouse model can be used to study mechanisms of cell death but the
disadvantages are that it requires intracerebral injection and Alpha synuclein inclusions are not
present. MPTP model is useful to study the mechanisms of cell death as well as screen therapies
that may improve the symptoms of PD. It produces a loss of Dopaminergic neurons and reduced
dopamine levels in the striatum. However, the motor impairments are less obvious in rodent
models of acute dosage of MPTP and Alpha synuclein inclusions are rare. Another disadvantage
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is that the cell death that occurs in this model is non-progressive. Paraquat and Rotenone models
are useful in testing neuroprotective strategies (Blesa et al. 2012).
A53T was the first missense mutation in the alpha synuclein gene that was associated with
autosomal – dominant PD and then missense mutations such as A30P, E46K etc. along with
multiplications of the SNCA locus are identified as genetic causes of PD. Genome wide
association studies have shown a link between the Alpha synuclein gene and sporadic AD as
well. Alpha synuclein mouse models therefore have high construct validity since they are based
on human genetic information regarding mutations or overexpression of the alpha synuclein gene
in PD (Koprich, Kalia and Brotchie 2017). The alpha synuclein mouse model that has the best
ability to reproduce the clinical and pathological features of the human disease is the human
Wild type Alpha Synuclein under the control of the Thy- 1 promoter which is one of the
proposed models for this study (Rockenstein et al. 2002). On the whole Alpha synuclein models
exhibit key features such as alpha synuclein inclusions, nigrostriatal dysfunction, motor and non-
motor phenotypes (Koprich et al. 2017) but there is no dopaminergic neuron death observed in
most Alpha Synuclein models (Blesa et al. 2012).
Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause a dominant form of PD but
LRRK2 mouse models show a lack of degeneration and no Alpha synuclein aggregation. These
models are useful for studying the role of LRRK2 mutations related to PD. Knockout models of
genes like Parkin, DJ1 and PTEN-induced novel kinase 1 (PINK1) that cause autosomal
recessive Parkinson’s are some of the other genetic models available (Blesa et al. 2012).
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3.5 Diet and PD models
As dietary restriction (DR) has been found to extend lifespan and reduce oxidative stress
levels, mice were treated with either a DR regimen for 3 months or DR mimic 2-deoxy-D-
glucose (2-DG), a non-metabolizable analogue of glucose for 7 days or before an acute dose of
MPTP injections. Mice on 2-DG or DR showed a reduction in the damage of dopaminergic
neurons and improved behavior in the rotary rod. Heat shock protein-70 (HSP-70) and glucose
regulated protein-78 (GRP-78) levels were increased in cultured dopaminergic cells (SK-N-MC)
pre-treated with 2-DG and in the striatum of 2-DG and DR treated mice. Cultured dopaminergic
cells pre-treated with 2-DG for 24 hours prevented the cell death induced by Rotenone, a
complex 1 inhibitor but the cells were not protected when treated immediately prior to Rotenone
exposure (Duan and Mattson 1999). Adult male rhesus monkeys on a 30% calorie restriction diet
prior to MPTP treatment had improved motor function, higher levels of dopamine and its
metabolites in the striatum along with an increased survival of dopaminergic neurons in
substantia nigra compared to rhesus monkeys on the regular diet. The CR monkeys also had
significantly higher levels of glial derived neurotropic factor (GDNF) in the caudate nucleus
compared to the controls (Maswood et al. 2004). GDNF is known to have neuro restorative and
neuroprotective properties and can protect dopaminergic cells against MPTP toxicity or a
metabolic insult (Gash et al. 1996, Hanbury et al. 2003).
Mice expressing mutant (A53T) α-synuclein that causes familial PD, have an elevated
resting heart rate characteristic of aberrant regulation by the autonomic nervous system. This was
associated with alpha synuclein aggregation in the brainstem and a reduction in para
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sympathethic activity. When the mice were given a high fat diet, the ANS abnormality was
exacerbated, while mice in the IF group it was reversed (Griffioen et al. 2013).
In a 6-Hydroxy dopamine (6-OHDA) rat model of Parkinson’s disease, dietary restriction
given two or 8 weeks before 6-OHDA treatment did not prevent nigrostriatal degeneration. The
authors proposed that this outcome is due to a short period of dietary restriction before 6-OHDA
lesioning which did not give sufficient time for the activation of stress responses (Armentero et
al. 2008).
A recent study has shown that intermittent fasting (IF) exacerbated dopamine neuron
degeneration in 8-week-old male mice administered Rotenone for 28 days. This is the first study
that investigated the effects of IF in mice when the dietary intervention was given along with and
not prior to Rotenone treatment. Mice in the IF treatment group treated with Rotenone had a
decrease in TH+ neurons, increase in alpha synuclein accumulation in the substantia nigra (SN)
and an increased motor dysfunction as demonstrated by a higher number of falls in rotarod est
compared to mice treated with Rotenone on regular chow and control mice on regular chow/IF.
The authors found that this effect of IF was due to an increase in inflammatory phospholipids
such as lysophospholipids and sphingomyelin and excitatory amino acids like glutamate,
aspartate, glycine and glutamine in the SN (Tatulli et al. 2018).
A diet that mimics the effects of fasting on markers associated with the stress resistance
caused by periodic fasting such as low levels of IGF-1, was identified by the Longo Lab. Fasting
Mimicking Diet (FMD) cycles extended longevity in mice given cycles of FMD starting at
middle age (Brandhorst et al. 2015). Previous research from the Longo lab has shown that fasting
mimicking diets can be used to prevent or slow the progression of a number of diseases namely-
65
diabetes, multiple sclerosis, cancer, Inflammatory Bowel disease. (Rangan et al. 2019, Choi et al.
2016, Cheng et al. 2017, Di Biase et al. 2016).
Periodic Fasting cycles lasting two or more days separated by at least a week of normal
diet can protect normal cells from a number of toxins, decrease blood glucose, insulin, and IGF-1
(Lee et al. 2010) while increasing autophagy (Brandhorst et al. 2015).Periodic fasting causes a
major reduction in the levels of white blood cells followed by stem cell based immune
regeneration upon refeeding (Cheng et al. 2014). However, it is difficult for majority of the
population to comply with prolonged water only fasting. It could have adverse effects and
exacerbate previous malnourishments especially in frail subjects (Brandhorst et al. 2015).
Fasting Mimicking diets were developed first for mice and then for humans to address these
concerns. The human FMD consists of a 5 day regimen providing between 725 and 1090 kilo
calories, with a macronutrient content selected to mimic water only fasting and a micronutrient
content to maximize nourishment (Longo and Panda 2016). In a recent study, FMD has been
found to be feasible in reducing disease risk factors in individuals from 20 to 70 years of age
(Wei et al. 2017).
The effects of Alternate Day Fasting (ADF) and Calorie restriction (CR) have mainly been
studied in neurotoxin models of Parkinson’s disease (MPTP, 6-OHDA, Rotenone) and mutant
Alpha Synuclein models. As mentioned above, there have been contradictory results on the
effects of dietary restriction in models of PD depending on the type of diet (30 % Calorie
Restriction or Alternate Day fasting), PD model used, the duration of the diet, and when the diet
was administered to the animals or dopaminergic cells (Duan and Mattson 1999, Maswood et al.
2004, Griffioen et al. 2013, Armentero et al. 2008, Tatulli et al. 2018).Although a number of
66
studies have reported beneficial effects of ADF and protection against different diseases, there
have been some reports of adverse effects such as impaired glucose tolerance(Tatulli et al. 2018,
Mattson, Longo and Harvie 2017). Alternate day fasting in Parkinson’s disease mouse models
usually consisted of 24 hours of feeding (ad libitum) followed by a day of water only fasting
(Duan and Mattson 1999, Tatulli et al. 2018). In a recent study, mice with Inflammatory Bowel
Disease(IBD) were given cycles of FMD or were on water-only fasting cycles for two days
followed by 12 days of refeeding . The combination of fasting with certain ingredients (FMD)
was effective in the mouse model of IBD but water-only fasting cycles was not sufficient in
reversing the symptoms of IBD (Rangan et al. 2019). In studies where dietary restriction showed
a protective function in Parkinson’s disease, it was usually applied for months before the
neurotoxic insult, allowing the activation of neuronal stress responses that could mitigate the
neurodegenerative process such as enhancing the production of neurotrophic factors (i.e., glial
cell line-derived neurotrophic factor, brain-derived neurotrophic factor) (Duan and Mattson
1999, Maswood et al. 2004). FMD promotes neurogenesis by the downregulation of IGF-1 and
PKA signaling, indicating that the generation of new and functional neurons may contribute to
the enhanced cognitive performance and motor co-ordination in old FMD-treated mice. An age-
dependent increase in muscle p62 was observed in 20-month-old mice fed a regular diet , but not
in the FMD groups indicating that the FMD protects muscle cells from age-dependent functional
decline which includes the ability to maintain normal expression of autophagy proteins
(Brandhorst et al. 2015). FMD reduced markers of inflammation, inhibited mTOR and PKA in
mouse models of diabetes (Cheng et al. 2017) and reduced pro- inflammatory cytokines (TNFα
and IFNγ levels) and microglia in a Multiple Sclerosis mouse Model (Choi et al. 2016). FMD
67
cycles reduced intestinal inflammation, stimulated protective gut microbiota, and reversed
intestinal pathology induced by chronic Dextran sodium sulfate in mice (Rangan et al. 2019).
In a study by Sampson et al. 2016, gut microbiota was found to influence Alpha
Synuclein-mediated neuroinflammation and motor deficits in the Alpha Synuclein Over-
expressing (ASO) mouse model of Parkinson’s disease. Microbiota from PD patients resulted in
an exacerbation of the motor deficits, Alpha Synuclein aggregation and an increase in neuro
inflammation. Germ free mice had a reduction in alpha synuclein accumulation, reduced neuro
inflammation and no motor deficits (Sampson et al. 2016). Thus, the modulation of the
microbiome can result in changes in Alpha Synuclein aggregation and neuro inflammation in the
ASO mouse model of Parkinson’s disease.
Previous research in the Longo lab has shown that FMD cycles have proven to have anti-
inflammatory effects in mouse models(reduction in inflammatory cells and cytokines) of auto-
immune disorders (Choi et al. 2016), improve motor co-ordination (Brandhorst et al. 2015) ,
influence favorable microbiota (Rangan et al. 2019), inhibit mTOR which is important in the
clearance of Alpha Synuclein (Kim et al. 2015) . Three one-week cycles of FMD, two cycles
before and one cycle after MPTP administration, improved motor co-ordination, prevented the
loss of dopaminergic neurons, reduced microglia and the release of pro-inflammatory cytokines
through the modulation of the microbiome. This study showed a neuroprotective role of short-
term FMD cycles on an acute neurotoxic mouse model of Parkinson’s disease(Zhou et al. 2019).
As the effect of FMD on the clearance of Alpha Synuclein oligomers/ aggregates was not
investigated previously and FMD was administered before the loss of motor co-ordination rather
that when the motor deficits already develop(Zhou et al. 2019), we investigated the effects of bi-
68
monthly cycles of FMD for ~6.5 or ~12.5 months starting at 2 months of age in a genetic mouse
model of Parkinson’s disease- a human Alpha Synuclein Overexpressing (ASO) mouse model
that has progressive motor deficits and alpha synuclein pathology of this disease(Fleming et al.
2004, Chesselet et al. 2012).
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3.6 Hypothesis and Research Design
The main hypothesis of this study is that the fasting mimicking diet can delay the progression
of motor deficits in a mouse models of Parkinson’s disease through a reduction in Alpha Synuclein
accumulation, neuroinflammation, microglial activation and an increase in mitochondrial
biogenesis.
Here, we demonstrate that 4-day FMD cycles, administered bi-monthly in genetic mouse model
of Parkinson’s disease - a) delayed the progression of motor deficits over time as assessed by the
spontaneous activity in a cylinder test, b) showed a trend for a reduction in markers of
neuroinflammation and activated microglia in the mid brain and c) showed a trend in the size of
Proteinase K resistant Alpha Synuclein aggregates in the substantia nigra.
To study the effects of FMD on the progression of motor deficits and PD-related pathology in
an ASO mouse model of Parkinson’s disease, FMD cycles were administered to the mice at 2
months of age for a period of ~6.5 or 12.5 months of age. At 2 months of age, the ASO mice
already develop motor deficits which become progressively worse with age. Thus, the end points
of the study were at ~8.5 months and ~14.5 months of age. At ~8.5 months, Alpha Synuclein
pathology is more severe compared to previous months and at 14 months of age, the ASO mice
show loss of striatal dopamine.
Motor co-ordination was measured at baseline (2 months) before FMD was administered, and
at 4, 6.5 and 8.5 months of age using the test of spontaneous activity in a cylinder and the
challenging beam test. There was an increase in number of forelimb and hindlimb steps in a test
of spontaneous activity in the cylinder in ASO FMD mice compared to ASO Control mice. In
order to determine if FMD cycles had a neuroprotective effect on motor deficits with respect to
fine motor skills, a separate cohort of mice were given FMD cycles starting at 1 month of age for
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~7.5 months. At 8.5 months of age, the mice were assessed in their ability to build a nest which
tests their fine motor skills.
We assessed mRNA levels of IL1β, CD68 and PGC-1α from RNA extracted from the mid-
brain of ~14.5-month-old mice and found that levels of IL1β and CD68 were reduced in the mid-
brain of ASO FMD mice compared to ASO Control. The number of Proteinase K Resistant Alpha
Synuclein aggregates in14.5-month-old mice was assessed by staining sections of the brain
containing the substantia nigra treated with proteinase K for human and mouse Alpha synuclein.
FMD cycles reduced the number of Alpha Synuclein aggregates that were greater than 20 microns.
Thus, we show that FMD cycles containing low protein, low carbohydrates and higher
unsaturated fat containing have the potential to delay the progression of motor deficits, reduce
neuroinflammation and microglial activation, and reduce the size of Alpha Synuclein aggregates.
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Chapter 4: The impact of Fasting Mimicking Diet cycles on motor-coordination, Alpha
Synuclein aggregates, neuroinflammation and microglial activation in an Alpha Synuclein
overexpression mouse model of Parkinson’s disease.
4.1 Abstract
Dietary Interventions such as FMD cycles are potential treatments for neurodegenerative
diseases. In this study, we demonstrate that FMD cycles can improve motor co-ordination in an
Alpha Synuclein Overexpressing (ASO) mouse model of Parkinson’s disease when the diet is
administered even when the motor deficits have already begun.
In male ASO mice that were given FMD cycles for ~12.5 months of age starting at 2
months and showed an increase in motor-coordination at 8.5 months of age, this improvement was
also accompanied by a reduction in markers related to neuroinflammation and microglial
activation but not mitochondrial biogenesis in the midbrain of the mice. In ~8.5-month-old mice
that were administered FMD for ~6.5 months and were euthanized 7 days post refeeding, serum
IGF-1 levels remain unchanged across all the four groups- WT Control, WT FMD, ASO Control
and ASO FMD. IGF-1 levels in the serum of ~14.5-month-old ASO FMD mice were similar to
ASO Control mice but were significantly lower compared to WT Control mice indicating that
FMD cycles are unable to further reduce IGF-1 levels at 14.5 months of age when there is also a
loss of striatal dopamine. We also observed a reduction in the number of Proteinase K resistant
Alpha Synuclein aggregates that were greater than 20 microns in the Substantia Nigra (SN) of
~14.5 month old FMD-treated ASO mice.
The findings from this study indicate that FMD cycles have an effect on neuroinflammation,
microglial activation and Alpha Synuclein pathology resulting in the improvement of motor co-
ordination in ASO mice, and thus is a potential treatment for Parkinson’s disease.
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4.2 Results
Bi-monthly FMD cycles increase the forelimb steps in a behavior test of spontaneous activity
in a cylinder in male ASO and WT mice and show a trend towards an increase in the
hindlimb steps in a behavior test of spontaneous activity in male ASO and WT mice.
Through the use of the Alpha Synuclein overexpressing mouse model (ASO) under the
control of the murine Thy-1 promoter (Rockenstein et al. 2002, Fleming et al. 2004, Chesselet et
al. 2012), we determined whether cycles of the fasting mimicking diet (FMD) starting at 2 months
for a duration of ~6.5 months could slow the progression of the motor deficits as evaluated by the
spontaneous activity in a cylinder and the challenging beam test. FMD was administered for 4 days
followed by 10 days of refeeding with the control diet. The cycles of FMD were bi-monthly. The
Human Prolon FMD is composed of 44% Carbohydrates, 9 % Protein and 47% Fat compared to
the control diet that has a composition of 64% Carbohydrates, 19 % Protein and 17% Fat
(Schematic 3). After 4 days on the FMD, the ASO as well at the WT mice lose 15-20 % of their
body weight but close to 100 % of their body weight is regained one day after refeeding with
regular chow (data not shown).
Schematic 3. Percentage of macronutrients in Human Prolon versus the Control diet
10 days refeed 4 days diet FMD – Human ProLon
Human Prolon FMD
Protein ~ 9%
Carb ~ 44%
Fat ~ 47%
Control Diet (AIN93)
Protein~19%
Carb ~ 64%
Fat ~17%
9%
44%
47%
Pro.
Carb.
Fat
19%
64%
17%
Prot.
Carb.
Fat
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Motor co-ordination was measured by spontaneous activity in a cylinder and the
challenging beam test at baseline (2 months), 4, 6.5 and 8.5 months of age in WT Control, WT
FMD, ASO Control and ASO FMD mice. Mice were euthanized at 14.5-15 months of age, 7 days
post refeeding after the last cycle of FMD (Figure 17B). Although the end point for behavior was
conducted at 8.5 months of age, the mice were administered FMD cycles till 14.5-15 months of
age (for a total of ~ 12.5 to 13 months) to assess Alpha Synuclein pathology and other markers of
neuroinflammation, after loss of dopamine in the striatum that occurs after 14 months of age(Lam
et al. 2011) . Percentage survival of the mice was determined at 14.5-15 months of age. FMD
cycles did not affect the percent survival of WT mice and there was no significant difference in
the survival between ASO FMD and ASO Control groups. However, WT Control as well as WT
FMD mice had a non-significant trend towards a higher percent survival compared to ASO FMD
mice (p=0.0902, WT Control versus ASO FMD; p=0.0782, WT FMD versus ASO FMD; Figure
17B).
0 5 10 15 20
0
20
40
60
80
100
Age (Months)
Percent survival
ASO and WT Males
WT Control (n=20)
WT FMD (n=21)
ASO FMD (n=23)
ASO Control (n=22)
10 days
2 months
14.5 months
Tissue collection at 14.5-15 months
7 days after refeeding
FMD
1 cycle
14 days Control Diet
ASO / WT Males
4 days
4 months
6.5 months
8.5 months
9 months
A
B
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Figure 17. Experimental timeline and percent survival in ASO and WT male mice.
(A) Experimental diet and behavior schedule for ASO and WT males starting at 2 months of age through 14.5 months of age.
(B) Kaplan-Meier survival curves for WT Control (n=19/20), WT FMD (n=20/21), ASO Control (n=15/22) and ASO FMD
(n=8/23). Kruskal-Wallis test.
The ASO mice have shown to have motor deficits as assessed by the challenging beam and
the test of spontaneous activity in the cylinder that begin at 2 months of age and become
progressively worse with age(Fleming et al. 2004). We measured the forelimb steps and
hindlimb steps of male WT Control, WT FMD, ASO Control and ASO FMD at 2, 4, 6.5 and 8.5
months of age in a test of spontaneous activity in a cylinder (Figure 18 and 19). Comparison of
WT and ASO mice (before dietary intervention) at 2 months (baseline), showed that the forelimb
steps in WT mice were significantly higher than ASO mice (*p<0.05; Figure 18A). ASO mice at
baseline (before assigned to the different diet groups) did not show a significant difference in the
forelimb steps and this indicates that the interpretation of the results of forelimb steps in the
following months cannot be attributed to a difference in the groups before the dietary
intervention (Figure 18B). There is a significant difference in forelimb steps across all the four
groups over time using 2-way ANOVA (**p<0.01; Figure 18C). ASO FMD mice take
significantly more forelimb steps in the cylinder compared to ASO Control mice (*p<0.05;
Figure 18D). WT Control mice show a significant increase in forelimb steps compared to ASO
Control mice (***p<0.001; Figure 18E) which is similar to what is previously published
(Fleming et al. 2004). WT Control and WT FMD do not show any significant differences in
forelimb steps (Figure 18F).
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Figure 18. FMD cycles increase the forelimb steps in a behavior test of spontaneous activity
in a cylinder in male ASO and WT mice.
(A) Forelimb steps at baseline (2 months of age) between ASO (n=29) and WT males(n=28).
(B) Forelimb steps at baseline (2 months of age) between ASO Control (n=17) and ASO FMD males(n=12).
(C) Comparison of forelimb steps between WT control (n=16), WT FMD (n=11-12), ASO Control (n=13-17) and ASO FMD (n=9-
12) at 2, 4, 6.5 and 8.5 months of age.
(D) Forelimb steps between ASO Control (n=13-17) and ASO FMD (n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(E) Forelimb steps between WT Control (n=16) and ASO Control (n=13-17) at 2, 4, 6.5 and 8.5 months of age.
(F) Forelimb steps between WT Control (n=16) and WT FMD (n=11-12) at 2, 4, 6.5 and 8.5 months of age.
Data are presented as mean ± SEM.
For Figure 18A-B: ∗p < 0.05, Unpaired 2-tailed student’s t-test.
For Figure 18C-F: ∗p < 0.05, ∗∗p < 0.01***p < 0.001, Two-way ANOVA.
A B
C D
E F
0 2 4 6 8 10
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
ASO FMD (n=9-12)
WT FMD (n=11-12)
ASO Control (n=13-17)
WT Control (n=16)
**p<0.01
0 2 4 6 8 10
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
ASO FMD (n=9-12)
ASO Control(n=13-17)
* p<0.05
0 2 4 6 8 10
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
WT FMD(n=11-12)
WT Control (n=16)
0 2 4
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
Forelimb steps at Baseline (ASO)
ASO FMD (n=12)
ASO Control (n=17)
0 2 4 6 8 10
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
ASO Control(n=12-17)
WT Control (n=16)
*** p<0.001
0 2 4
0
20
40
60
80
100
120
140
time (months)
Forelimb steps
ASO Control (n=29)
WT Control (n=28)
Forelimb steps at Baseline
*
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The number of Hindlimb steps in the cylinder are significantly different between male ASO
and WT mice even at 2 months of age with ASO mice taking much fewer hindlimb steps
compared to WT mice(Fleming et al. 2004, Fleming, Ekhator and Ghisays 2013). Comparison of
male WT and ASO mice (before dietary intervention) at 2 months (baseline), showed that the
hindlimb steps in WT mice were significantly higher than ASO mice (****p<0.0001; Figure
19A). There was no significant difference between ASO mice at baseline in the number of
hindlimb steps (Figure 19B). There is a significant difference in hindlimb steps across all the
four groups over time using 2-way ANOVA (****p<0.0001; Figure 19C). ASO FMD mice show
a trend towards significance in taking a higher number of hindlimb steps in the cylinder
compared to ASO Control mice (p=0.0863; Figure 19D). WT Control mice show a significant
increase in hindlimb steps compared to ASO Control mice across all the time points
(****p<0.0001; Figure 19E), which is similar to what is previously published (Fleming et al.
2004). There is no significant difference between WT Control and WT FMD in hindlimb steps
(Figure 19F).
These results indicate that FMD cycles for ~6.5 months in male ASO mice starting the diet
at 2 months of age (when the motor deficits are already present), can improve motor co-
ordination by increasing the number of forelimb and hindlimb steps in a cylinder.
77
Figure 19. FMD cycles show a trend towards an increase in the hindlimb steps in a behavior
test of spontaneous activity in male ASO and WT mice.
(A) Hindlimb steps at baseline (2 months of age) between ASO (n=29) and WT males(n=28).
(B) Hindlimb steps at baseline (2 months of age) between ASO Control (n=17) and ASO FMD males(n=12).
(C) Comparison of Hind limbs steps between WT control (n=16), WT FMD (n=11-12), ASO Control (n=13-17) and ASO FMD
(n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(D) Hindlimb steps between ASO Control (n=13-17) and ASO FMD (n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(E) Hindlimb steps between WT Control (n=16) and ASO Control (n=13-17) at 2, 4, 6.5 and 8.5 months of age.
(F) Hindlimb steps between WT Control (n=16) and WT FMD (n=11-12) at 2, 4, 6.5 and 8.5 months of age.
Data are presented as mean ± SEM.
For Figure 19A-B: ****p < 0.0001, Unpaired 2-tailed student’s t-test.
For Figure 19C-F: ****p < 0.0001, Two-way ANOVA.
A B
C D
E F
0 2 4 6 8 10
0
20
40
60
80
time (months)
Hind limb steps
ASO FMD (n=9-12)
WT Control (n=16)
WT FMD (n=11-12)
ASO Control (n=13-17)
****p<0.0001
0 2 4 6 8 10
0
20
40
60
80
time (months)
Hind limb steps
WT Control (n=16)
WT FMD (n=11-12)
0 2 4 6 8
0
10
20
30
time (months)
Hind limb steps
ASO FMD (n=9-12)
ASO Control (n=13-17)
p=0.0863
0 2 4 6 8
0
20
40
60
80
Hindlimb steps at Baseline
time (months)
Hind limb steps
WT Control (n=28)
ASO Control (n=29)
****
0 2 4 6 8 10
15
20
25
30
time (months)
Hind limb steps
ASO FMD (n=12)
ASO Control (n=17)
Hindlimb steps at Baseline (ASO)
0 2 4 6 8 10
0
20
40
60
80
time (months)
Hind limb steps
WT Control(n=16)
ASO Control (n=13-17)
****p<0.0001
78
Bi-monthly FMD cycles do not increase the number of rears and grooming time in a behavior
test of spontaneous activity in male ASO and WT mice.
We also measured the number of rears and grooming of male WT Control, WT FMD, ASO
Control and ASO FMD at 2, 4, 6.5 and 8.5 months of age in a test of spontaneous activity in a
cylinder (Figure 20 and 21). Before dietary intervention at 2 months (baseline), the number of rears
in WT mice were not significantly different compared to ASO mice (Figure 20A). ASO mice at
baseline (before assigned to the different diet groups) did not show a significant difference in the
number of rears (Figure 20B). There is no significant difference in the number of rears across all
the four groups over time using 2-way ANOVA (Figure 20C) and also between WT Control and
ASO Control over the different time points (Figure 20E). We did not observe any significant
difference in the number of rears between ASO Control and ASO FMD groups (Figure 20D) and
between WT Control and WT FMD (Figure 20F).
At 2 months (baseline), grooming time in WT mice was significantly different compared to
ASO mice (***p<0.001; Figure 21A). ASO mice at 2 months before dietary intervention, did not
show a significant difference in grooming time (Figure 21B). There is no significant difference in
the number of rears across all the four groups over time using 2-way ANOVA (Figure 21C) but
the grooming time in WT Control mice was significantly higher compared to ASO Control mice
over the different time points (***p<0.001; Figure 21E). There was no significant difference in
the grooming time between ASO Control and ASO FMD groups (Figure 21D) and between WT
Control and WT FMD (Figure 21F).
79
Figure 20. FMD cycles do not increase the number of rears in a behavior test of spontaneous
activity in male ASO and WT mice.
(A) The number of rears at baseline (2 months of age) between ASO (n=29) and WT males(n=28).
(B) The number of rears at baseline (2 months of age) between ASO Control (n=17) and ASO FMD males(n=12).
(C) Comparison of the number of rears between WT control (n=16), WT FMD (n=11-12), ASO Control (n=13-17) and ASO FMD
(n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(D) The number of rears between ASO Control (n=13-17) and ASO FMD (n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(E) The number of rears between WT Control (n=16) and ASO Control (n=13-17) at 2, 4, 6.5 and 8.5 months of age.
(F) The number of rears between WT Control (n=16) and WT FMD (n=11-12) at 2, 4, 6.5 and 8.5 months of age.
Data are presented as mean ± SEM.
For Figure 20A-B: Unpaired 2-tailed student’s t-test.
For Figure 20C-F: Two-way ANOVA.
A
0 2 4 6 8 10
0
5
10
15
20
25
time (months)
Number of Rears
WT Control (n=16)
WT FMD (n=11-12)
ASO Control (n=13-17)
ASO FMD (n=9-12)
B
0 2 4 6 8 10
0
5
10
15
20
time (months)
Number of Rears
ASO Control (n=13-17)
ASO FMD (n=9-12)
C D
E F
0 2 4
12
14
16
18
time (months)
Number of Rears
Rears at Baseline
WT (n=28)
ASO (n=29)
0 2 4
10
12
14
16
18
time (months)
Number of Rears
Rears at Baseline (ASO)
ASO Control (n=17)
ASO FMD (n=12)
0 2 4 6 8 10
0
5
10
15
20
25
time (months)
Number of Rears
WT Control(n=16)
WT FMD (n=11-12)
0 2 4 6 8 10
0
5
10
15
20
time (months)
Number of Rears
WT Control(n=16)
ASO Control(n=13-17)
80
Figure 21. FMD cycles do not increase grooming time in a behavior test of spontaneous
activity in male ASO and WT mice.
(A) Grooming time at baseline (2 months of age) between ASO (n=29) and WT males(n=28).
(B) Grooming time at baseline (2 months of age) between ASO Control (n=17) and ASO FMD males(n=12).
(C) Comparison of grooming time between WT control (n=16), WT FMD (n=11-12), ASO Control (n=13-17) and ASO FMD
(n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(D) Grooming time between ASO Control (n=13-17) and ASO FMD (n=9-12) at 2, 4, 6.5 and 8.5 months of age.
(E) Grooming time between WT Control (n=16) and ASO Control (n=13-17) at 2, 4, 6.5 and 8.5 months of age.
(F) Grooming time between WT Control (n=16) and WT FMD (n=11-12) at 2, 4, 6.5 and 8.5 months of age.
Data are presented as mean ± SEM.
For Figure 21A-B: ***p < 0.001, Unpaired 2-tailed student’s t-test.
For Figure 21C-F: ***p < 0.001, Two-way ANOVA.
A
0 2 4
0
10
20
30
40
Grooming time at baseline
time (months)
Grooming time (seconds)
WT (n=28)
ASO (n=29)
***
0 2 4
0
10
20
30
40
Grooming time at baseline (ASO)
time (months)
Grooming time (seconds)
ASO Control (n=17)
ASO FMD (n=12)
B
0 2 4 6 8 10
0
10
20
30
40
time (months)
Grooming time (seconds)
WT Control (n=16)
ASO Control (n=13-17)
*** p<0.001
E
0 2 4 6 8 10
0
5
10
15
20
time (months)
Grooming time (seconds)
ASO Control(n=13-17)
ASO FMD (n=9-12)
0 2 4 6 8 10
0
10
20
30
40
50
time (months)
Grooming time (seconds)
WT Control (n=16)
WT FMD (n=11-12)
F
D C
0 2 4 6 8 10
0
10
20
30
40
50
time (months)
Grooming time (seconds)
WT Control (n=16)
WT FMD (n=11-12)
ASO Control (n=13-17)
ASO FMD (n=12)
81
Time to traverse across a challenging beam in male ASO and WT mice on either FMD or a
Control diet.
In the same group of mice, we also assessed motor co-ordination in a challenging beam test
where mice were trained to walk a beam for two days consisting of segments with varying width
and on the third day, a wire mesh was placed above the beam to increase the level of difficulty and
as well as determine the fine motor skills of the mice to be able to grasp the mesh (Fleming et al.
2004, Fleming et al. 2013). At 2 months (baseline), time to traverse the beam in WT mice was not
significantly different compared to ASO mice (Figure 22A). ASO mice at 2 months (baseline), did
not show a significant difference in time to traverse the challenging beam (Figure 22B). There was
a significant difference in the time to traverse across all the four groups over time using 2-way
ANOVA (****p<0.0001; Figure 22C) and the time to traverse of WT Control mice was
significantly lower compared to ASO Control mice over the different time points (****p<0.0001;
Figure 22E). There was no significant difference in the time to traverse between ASO Control and
ASO FMD groups (Figure 22D) and between WT Control and WT FMD (Figure 22F).
82
Figure 22. Time to traverse across a challenging beam in male ASO and WT mice on either
FMD or a Control diet.
(A) Time to traverse at baseline (2 months of age) between ASO (n=34) and WT males(n=33).
(B) Time to traverse at baseline (2 months of age) between ASO Control (n=20) and ASO FMD males(n=14).
(C) Comparison of Time to traverse between WT control (n=16-18), WT FMD (n=9-15), ASO Control (n=10-20) and ASO FMD
(n=13-14) at 2, 4, 6.5 and 8.5 months of age.
(D) Time to traverse between ASO Control (n=10-20) and ASO FMD (n=13-14) at 2, 4, 6.5 and 8.5 months of age.
(E) Time to traverse between WT Control (n=16-18) and ASO Control (n=10-20) at 2, 4, 6.5 and 8.5 months of age.
(F) Time to traverse between WT Control (n=16-18) and WT FMD (n=9-15) at 2, 4, 6.5 and 8.5 months of age.
Data are presented as mean ± SEM.
For Figure 22A-B: Unpaired 2-tailed student’s t-test.
For Figure 22C-F: ****p < 0.001, Two-way ANOVA.
A B
C D
E F
0 2 4 6 8 10
0
5
10
15
20
time (months)
Time to traverse in seconds
WT Control (n=16-18)
ASO Control (n=10-20)
****p<0.0001
0 2 4 6 8 10
0
5
10
15
20
time (months)
Time to traverse in seconds
ASO Control (n=10-20)
ASO FMD (n=13-14)
0 2 4 6 8 10
0
5
10
15
20
time (months)
Time to traverse in seconds
WT Control (n=16-18)
WT FMD (n=9-15)
ASO Control (n=10-20)
ASO FMD (n=13-14)
****p<0.0001
0 2 4 6 8 10
0
5
10
15
20
time (months)
Time to traverse in seconds
WT Control (n=16-18)
WT FMD (n=9-15)
0 2 4
10
12
14
16
18
time (months)
Time to traverse in seconds
ASO Control (n=20)
ASO FMD (n=14)
0 2 4
10
11
12
13
14
15
16
time (months)
Time to traverse in seconds
WT Control (n=33
ASO Control (n=34)
83
Fine motor skills (nest building behavior) in 8.5-month-old male ASO and WT mice on either
FMD or a Control diet.
In order to determine whether FMD cycles could improve motor co-ordination when
administered at 1 month of age (before motor deficits are observed), we conducted a test of fine
motor skills (nest building behavior) on ASO and WT at ~8.5 months of age that began the diet
at 1 month. Nest building behavior was assessed by the nesting score by Deacon et al. 2006.
There was a reduction in the nesting score of the ASO control group compared to ASO FMD,
WT Control and WT FMD.
Figure 23. Fine motor skills (nest building behavior) in 8.5 month old male ASO and WT
mice on either FMD or a Control diet.
Nesting score between WT control (n=14), WT FMD (n=9), ASO Control (n=4) and ASO FMD (n=8) at 8.5 months of age.
Data are presented as mean ± SEM. Kruskal-Wallis test.
WT Control
WT FMD
ASO Control
ASO FMD
0
1
2
3
4
5
Nesting score
Nest building behavior at 8.5 months
WT Control(n=14)
WT FMD (n=9)
ASO Control (n=4)
ASO FMD (n=8)
84
FMD cycles do not significantly alter IGF-1 levels in male ASO and WT mice at 9 months
of age but reduce IGF-1 levels in WT and ASO groups compared to WT control at 14.5
months of age.
FMD cycles were found to downregulate IGF-1 signaling in old mice (Brandhorst et al.
2015). Here, we wanted to investigate whether serum IGF-1 levels were changed in ASO and
WT mice administered FMD cycles starting at 2 months of age for 6.5 to 7 or 12.5 to 13 months
of age. Serum was collected at the end of the study 7 days post refeeding after the last FMD
cycle. There was no difference in serum IGF-1 (ng/ml) levels across WT Control, WT FMD,
ASO Control and ASO FMD at ~8.5 months of age (Figure 24A). At ~ 14.5 months of age,
serum IGF-1 levels of WT FMD, ASO Control and ASO FMD were significantly different from
WT Control mice (**p<0.01; Figure 24B). This shows that long term FMD cycles do not further
reduce IGF-1 levels in ASO mice 7 days post refeeding.
Figure 24. FMD cycles do not significantly alter IGF-1 levels in male ASO and WT mice at
9 months of age but reduce IGF-1 levels in WT and ASO groups compared to WT control at
14.5 months of age.
(A) Serum IGF-1 (ng/ml) levels of WT control (n=9), WT FMD (n=7), ASO Control (n=12) and ASO FMD (n=10) at 9 months of
age.
(B) Serum IGF-1 (ng/ml) levels of WT control (n=6), WT FMD (n=5), ASO Control (n=6) and ASO FMD (n=6) at 14.5 months
of age.
Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, One-way ANOVA followed by Tukey’s Multiple comparisons test.
FMD cycles reduce the expression of mRNA levels of genes associated with
neuroinflammation and microglial activation (IL1β and CD68) in the mid-brain from 14.5-
month-old ASO mice.
WT Control
WT FMD
ASO Control
ASO FMD
0
200
400
600
Serum IGF-1 at ~8.5 months
IGF-1 ng/ml
A B
WT Control
WT FMD
ASO Control
ASO FMD
0
200
400
600
Serum IGF1 at ~14.5 months
IGF-1 ng/ml
***p<0.001
** comapred to WT Control
85
FMD has shown to reduce markers of inflammation, inhibit mTOR and PKA in mouse
models of diabetes (Cheng et al. 2017) and reduce pro-inflammatory cytokines (TNFα and IFNγ
levels) and microglia in a Multiple Sclerosis mouse Model(Choi et al. 2016). Young ASO mice
have microglial activation and an increase in pro-inflammatory cytokines such as tumor necrosis
factor-α (TNF-Alpha) in the striatum and substantia nigra. Microglial activation occurs first in the
striatum (1 month of age) and later in the substantia nigra (5-6 months of age) in the ASO mouse
model (Chesselet et al. 2012). Research from another lab on the effects of FMD in an acute MPTP
mouse model of Parkinson’s disease, showed that 3 one-week cycles of FMD administered before
as well as after a day of MPTP injections, reduced the number of IBA-1 positive microglia as well
as reduced the levels of pro-inflammatory cytokines -IL1β and TNFα (Zhou et al. 2019) .
We assessed the relative expression of genes such as IL1β, CD68 and PGC-1α from the
midbrain of ASO and WT mice on either FMD or Control diet at ~14.5 months of age (mice from
the experimental design in Figure 17A). PGC-1α, is a transcriptional coactivator that has emerged
as a master regulator of mitochondrial biogenesis (Curry et al. 2018) .
We observed a trend for a decrease in IL1β and CD68 mRNA levels in the mid brain of ASO
FMD mice compared to the other three groups- WT Control, ASO Control and WT FMD (Figure
25A and 25B). There was no trend towards a change in PGC-1α mRNA levels in the mid brain
across all the groups (Figure 25C).
86
Figure 25. FMD cycles reduce the expression of mRNA levels of genes associated with
neuroinflammation and microglial activation (IL1β and CD68) in the mid-brain from 14.5-
month-old ASO mice.
Relative expression of (A) IL1β, (B) CD68 (C) PGC-1α assessed by RT-qPCR from mRNA extracted from the mid-brain of WT
control, WT FMD, ASO Control and ASO FMD mice at 14.5 months of age. Data are presented as mean ± SEM and show fold
difference relative to the WT Control group. (n=2-3/group)
A
B
C
0
1
2
Relative Il-1 Beta Expression
ASO Control
WT Control
WT FMD
ASO FMD
0
2
4
Relative CD68 Expression
ASO Control
WT Control
WT FMD
ASO FMD
0
2
4
Relative PGC1 Alpha Expression
ASO Control
WT Control
WT FMD
ASO FMD
87
Proteinase K resistant Alpha Synuclein Aggregates in the Substantia of male ASO Control
versus ASO FMD mice at 14.5 months of age.
ASO mice have a loss of striatal dopamine at 14 months of age (Lam et al. 2011) and
Proteinase K resistant Alpha Synuclein aggregates of variable sizes are detected in the substantia
nigra of ASO mice at 5 months of age (Chesselet et al. 2012). Proteinase K resistant Alpha
Synuclein aggregates were observed in the Substantia Nigra of ~14.5-month-old ASO Control and
ASO FMD mice that were given the diet for ~12.5 months starting at 2 months of age (Figure
26A). Although there was no significant difference in the mean number of aggregates in the SN
between ASO Control and ASO FMD mice (Figure 26B), there was a reduction in the number of
aggregates in the SN that were greater than 20 microns in ASO FMD mice compared to ASO
Control mice (Figure 26C).
Figure 26. Proteinase K resistant Alpha Synuclein Aggregates in the Substantia of male ASO
Control versus ASO FMD mice at 14.5 months of age.
(A) Representative images showing Proteinase K Resistant Alpha Synuclein aggregates in the sections containing the substantia
nigra (SN) of 14.5-month-old male ASO Control and ASO FMD mice.
(B) Mean number of aggregates in the SN of male ASO Control versus ASO FMD mice (n=2 animals/group).
A
B C
ASO Control
ASO FMD
0
100
200
300
400
Mean number of aggregates in the SN
ASO Control (n=2)
ASO FMD (n=2)
0
5
10
Number of the aggregates
in the SN >20 microns
ASO Control (n=2)
ASO FMD (n=2)
88
(C) Number of Proteinase K resistant Alpha Synuclein aggregates greater than 20 microns in the SN of male ASO Control versus
ASO FMD mice (n=2 animals/group).
Data are presented as mean ± SEM.
Images were taken at 40x magnification.
Overall, these results show that long-term cycles of FMD in ASO mice starting at 2 months
of age (when the mice already exhibit motor impairments) increase the number of forelimb and
hindlimb steps in a test of spontaneous activity in a cylinder, modestly reduce the expression of
genes related to neuroinflammation and microglial activation and the size of Proteinase K
resistant Alpha Synuclein aggregates in the Substantia Nigra.
89
4.3 Discussion
PD has a complex etiology and there are currently no treatments to efficiently modify the
disease although there are promising candidates like the GLP-1R agonist, Exenatide (Lindholm et
al. 2016) . Previous treatments available can reduce the motor symptoms but not the non-motor
symptoms and some of them have adverse side effects (Lindholm et al. 2016, Stoker et al. 2018)
but the inhibition of mTOR by Rapamycin prevented the development of dyskinesia in mice given
L-DOPA (Santini et al. 2009). Neuroinflammation and proteostasis are important processes in the
development of the disease and the components of these pathways are potential targets for
therapy(Lindholm et al. 2016). Dietary intervention may enhance neuronal adaptive stress
responses and increase neurotrophic signaling, DNA repair, proteostasis and mitochondrial
biogenesis and can provide an important avenue for Parkinson’s disease risk reduction (Mattson
2014).
Results from the genetic mouse of Parkinson’s disease show that bi-monthly FMD cycles
for ~6.5 months starting at 2 months of age, improve motor co-ordination and delay the progression
of motor deficits at 8.5 months of age. We also show that that FMD cycles show a trend to reduce
the mRNA levels of genes related to neuroinflammation and microglial activation but not
mitochondrial biogenesis in the mid-brain. We observed that there was also a trend towards the
reduction in size of Proteinase K resistant Alpha Synuclein aggregates in the mid-brain of ASO
FMD mice. These results support that FMD cycles can delay the progression of motor deficits
through a modest reduction in neuroinflammation, microglial activation and Alpha Synuclein
pathology. We note that more mice should be added to our analysis for Alpha Synuclein pathology
and mRNA expression levels so that we can make a definite conclusion.
90
Utilizing the Alpha Synuclein Overexpressing model, a previous study showed that a
reduction in both Alpha Synuclein accumulation and in microglia activation was observed in germ
free ASO mice and this coincided with a reduction in motor deficits (Sampson et al. 2016).
Previous research has shown that Toll Like Receptor 2(TLR2) is elevated in patients with
Parkinson’s disease (Doorn et al. 2014). Oligomeric forms of extracellular Alpha Synuclein bind
to TLR2 on the surface of neuronal cells and microglia(Kim et al. 2013, Kim et al. 2015). Anti-
TLR2 alleviated α-synuclein accumulation in neuronal and astroglia cells, neuroinflammation,
neurodegeneration, and behavioral deficits in the ASO mouse model(Kim et al. 2018).Inhibition
of autophagy via the activation of AKT/mTOR by Alpha Synuclein binding to TLR2 results in
accumulation of α-synuclein oligomers and neuronal toxicity (Kim et al. 2015). Thus, we would
like to determine the levels of autophagy markers, other inflammatory cytokines and the
percentage of microglial and neuronal cells expressing TLR2, to identify the pathways in which
FMD cycles could confer stress resistance to a ASO mouse model. AMPK has been reported to
promote mitophagy by activating ULK1 and inhibiting (Zhang and Lin 2016) and promote
lysosomal biogenesis via increased TFEB activity (Curry et al. 2018).
AMPK plays an important role in mitochondrial biogenesis through the transcriptional and
post-translational activation of PGC-1α (Curry et al. 2018). PINK1 and PARKIN are essential
genes for PGC-1α signaling in which mutations cause autosomal-recessive Parkinson’s disease
(Lee et al. 2017, Wei et al. 2017). Parkin Q311X mice that have reduced levels of PGC-1α in the
striatum and Rapamycin could restore PGC-1α-TFEB signaling and mitochondrial quality control
(Siddiqui et al. 2015). In our study we did not find any change in PGC-1α mRNA levels in the
mid-brain of 14.5-month-old ASO FMD mice compared to ASO Control mice. However, as
mentioned previously, we will add more mice numbers to confirm the results. As the striatum of
91
the mice has also been collected, we can determine the mRNA levels of PGC-1α in ASO FMD
versus ASO Control mice at 14.5 months of age.
The behavior tests of motor co-ordination that were utilized in this study, helped to
characterize the motor deficits in the ASO mouse model and are highly sensitive to subtle changes
in nigrostriatal dysfunction (Fleming et al. 2004, Fleming et al. 2013). In the ASO mouse model,
motor deficits start to develop at 2 months and become progressively worse with age at around 8
months of age (Fleming et al. 2004) but without the loss of striatal dopamine occurs which is at 14
months of age (Lam et al. 2011). In our study, we started the administration of the diet 2 months
in order to determine if the progression of motor deficits could be delayed. In our future studies,
we would like to determine the effects of FMD, utilizing the Paraquat model of Parkinson’s disease
that also shows impairments in motor dysfunction along with the presence of dopaminergic cell
death (Chinta et al. 2018) and administering FMD cycles after Paraquat administration.
Thus, overall FMD cycles has potential in slowing the progression of Parkinson’s disease in a
genetic mouse model and we will continue to study its effects on neuroinflammation, microglial
activation, mitochondrial biogenesis as well as autophagy with respect to Alpha Synuclein
pathology.
92
4.5 Materials and Methods
Animal Models and Breeding Scheme- All animal protocols were approved by the Institutional
Animal Care and Use Committee (IACUC) of the University of Southern California. Transgenic
mice overexpressing Human wild- type Alpha Synuclein under the murine Thy-1 promoter were
created by (Rockenstein et al. 2002) and crossed in to a mixed C57BL/6 DBA2 background.
Since Alpha synuclein mice on C57BL/6 background are more prone to seizures, transgenic mice
were maintained on the mixed background by breeding Thy-1 Alpha Synuclein overexpressing
(ASO) females obtained from Dr. John Varghese’s laboratory at UCLA (under an MTA
agreement) with BDF1 (C57BL/6 DBA2) males from Charles River. This breeding scheme was
maintained since it has been previously reported that breeding wild- type (WT) females with
mutant males results in fewer progeny. The genotype of the Alpha Synuclein overexpressing
mice and the wild-type mice was verified by PCR analysis of ear piece or tail DNA. Animals
were maintained on a 12 hr light/dark cycle and were housed with littermates of the same sex
(maximum of four per cage) (Sampson et al. 2016, Fleming et al. 2004).
Body weight- Body weights (in grams) was measured a twice a week for all the WT and ASO
male mice on regular chow (ad libitum-fed), enrolled in the study. Body weight for WT and ASO
mice on FMD was measured every day during FMD (Day1-Day4), immediately after 4 days of
FMD (before refeeding), one day after refeeding and twice a week during the refeeding period.
Fasting Mimicking Diet- FMD (Human Prolon) was given to WT and ASO mice starting at 2
months of age for a period of either 6.5 months or 12.5 months. A separate group of mice were
given the FMD starting at 1 month of age for 7.5 months. Day 1 consisted of 50 % of normal
calorie intake and the diet on day 2-4 restricted the calorie intake to 30 % of the normal amount
93
(70% reduction in calories). ASO and WT mice lost 15- 20% body weight after day 4 on FMD
and were given regular chow on Day 5.
Behavior tests- All the behavior tests were conducted during the dark phase between 6 pm and
10 pm. Previous groups have published that the behavior tests of motor co-ordination on the
ASO mice have been conducted during 1 pm to 4 pm under low light but when the mice are
housed on a reverse light-dark cycle with the lights off at 10 am. Since it was not possible to
house the mice in a facility with the reverse light-dark cycle, the mice were tested during the
actual dark cycle as recommended by Fleming et al. 2013.
Challenging Beam- Motor co-ordination was measured using the challenging beam which was
constructed from Plexiglass and consisted of four sections (25 cm each, 1 m in length). Each
section had different widths ranging from 3.5 cm, 2.5 cm, 1.5 cm and 0.5 cm. The duration of the
test was three days and consisted of 5 trials per day. On the first and second day, mice were
trained to walk the beam starting from the widest section with the narrowest section leading
directly into the home cage. On the third day, to increase the level of difficulty a wire mesh of 1
cm squares and of corresponding width was placed over the beam surface. 1 cm width of
underhanging ledges were placed below each beam to provide a support when the mouse limb
slips through walking on the grid. Mice were videotaped during the third day while they walk the
grid-covered beam for 5 trials. The time taken to walk the grid-surfaced beam (time to traverse)
was noted when the video is viewed in real time. The time to traverse across all the 5 trials on the
test day were averaged for the wild type mice and the ASO mice (Fleming et al. 2004, Fleming et
al. 2013) . The mice were tested on the challenging beam at 2 months (baseline), 4 months, 6.5
months and 8.5 months of age (Fleming et al. 2004, Fleming et al. 2013) .
94
Spontaneous activity in a cylinder- Mice were placed in a transparent cylinder (height, 15.5 cm
and diameter 12.7 cm) for three minutes and videotaped. The number or rears, hindlimb steps,
forelimb steps and the time spent grooming was measured and compared between WT and ASO
mice on either ad libitum-fed control diet (regular chow) or FMD. A rear is described as a
vertical movement when only the hindlimbs of the mouse were on the surface of the glass placed
below the cylinder. A mirror is placed beneath the glass in a certain angle in order to assess the
number of steps and grooming time clearly on the surface and also along the walls of the
cylinder. An experimenter blind to the genotype and treatment group viewed the videotapes in
slow motion and counted the number of forelimb and hindlimb steps. The number of steps were
counted when the mouse moved both its hindlimbs or forelimbs across the glass surface
(Fleming et al. 2004). Grooming time was assessed by an experimenter viewing the video in real
time and included grooming bouts on the snout , vibrissae and the body (Fleming et al. 2013).
The mice were repeatedly tested for spontaneous activity in the cylinder at 2 months (baseline), 4
months, 6.5 months and 8.5 months of age as described previously (Fleming et al. 2004).
Nest building behavior - In order to assess fine motor skills in the ASO and WT mice on FMD
or on the control diet, nesting behavior was assessed at ~8.5 months of age. Mice were
individually housed in new cages during the duration of this test. A pre-weighed pressed cotton
square (nestlet) was placed in the cages of the mice and the nest was scored the next day from 1-
5 by the criteria outlined by Deacon 2006 as follows: 1 - 90 % or more of the nestlet was left
intact, 2 - 50-90% of the nestlet was intact, 3 – 50-90% of the nestlet has been shredded but there
was no noticeable nest, 4 – more than 90% of the nestlet was shredded but the nest is flat, 5- a
close to perfect nest with more than 90% of the nestlet shredded and the walls of the nest are
95
higher than the height of the mouse curled up on its side for greater than 50% of its
circumference (Deacon 2006).
Immunohistochemistry- Adult mice were anesthetized with isoflurane, punctured in the heart
for serum, followed by intracardial perfusion with saline. Brains were removed immediately and
cut in half. One half was post-fixed in 4% PFA for 48 hours and stored in 20% PB Sucrose
afterwards, while the other half was dissected for the cortex, hippocampus, cerebellum, mid
brain, brain stem (for all the mice in the study), striatum (only for 14.5-month-old mice) which
were frozen on dry ice and then stored at -80C until further processing. The hemi-brains were cut
in a coronal direction (40 μm) using a cryostat CM1850 (Leica) and stored in cryoprotectant
solution until ready for immunohistochemistry (IHC). Sections containing the SN were immuno-
stained in the subsequent protocol.
3,3’-diaminobenzidine (DAB) Staining - For 3,3’-diaminobenzidine (DAB)-based protocols,
sections were washed in 0.1M PBS, incubated in PBS containing 10 ug/ml proteinase-K
(Invitrogen) for 10 mins at room temperature, washed in 0.1M PBS, and incubated in 0.5% H202
in 0.1M PBS for 15 mins to block endogenous peroxidase activity. Sections were blocked in
Mouse on Mouse (MOM) blocking solution (Vector Laboratories) for 1 hour before incubating
with primary antibody- monoclonal mouse anti-α-synuclein (1:250; BD Biosciences, 610787,
formerly Transduction laboratories S63320), at 4° C in the presence of 2% normal goat serum
(NGS; Jackson ImmunoResearch) overnight. The following day, sections were washed in 0.1M
PBS, incubated with secondary antibody biotinylated goat anti-mouse IgG (H+L) (1:500; Vector
Laboratories, BA-9200) at room temperature for 2 hours in the presence of 2% normal goat
96
serum, ABC Vector Elite (Vector Laboratories), and DAB kits (Vector Laboratories) before
finally being mounted on slides and air-dried (Fleming et al. 2011).
Proteinase K resistant Alpha Synuclein Aggregates
Two sections from the substantia nigra for α- synuclein treated with proteinase K were used for
quantification of aggregates in ASO mice. As proteinase K-resistant aggregates of α-synuclein
are not observed in WT mice, quantification of Proteinase K resistant Alpha Synuclein
aggregates was carried out only on ASO mice (Fernagut et al. 2007).
Images of the SN were captured at 40X with the BZ-X710 All-in-One Fluorescence Microscope
(Keyence) and each image was converted into two 8-bit files using ImageJ (NIH). One binary
file was used to set the brightness and the contrast to visualize the inclusions and the second was
used to set the threshold manually and analyze the particles. Aggregates were defined by
circularity in order to exclude quantification of other artifacts. Image J was used to generate the
number of the proteinase K resistant Alpha Synuclein aggregates and percent area occupied by
aggregates (Fleming et al. 2011).
RNA isolation and quantitative PCR- Total RNA was isolated from the mid brain of ~14.5
month old ASO and WT mice with the RNeasy Lipid Tissue Mini Kit (Qiagen) and RNase-Free
DNase Set (Qiagen), according to manufacturer’s protocols. iScript cDNA synthesis system
(Bio-Rad) was used to reverse transcribe cDNA from 1 μg of purified RNA isolated from the
mid-brain of the ASO and WT mice. The cDNA was used to run real-time quantitative PCR with
SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a Bio-Rad CFX Connect
Thermocycler. The mid brain was analyzed for expression levels of cluster of differentiation 68
97
(CD68), IL1β and PGC-1α. The samples were run in duplicate and the PCR products were
normalized with corresponding expression levels of β-actin in the mid brain. The ΔΔ-CT method
was used to determine relative mRNA levels and the Cq value of the reference gene β-actin was
subtracted separately from the target genes. The resulting values were averaged, and fold
changes were calculated relative to the WT Control group as described by (Moser, Uchoa and
Pike 2018).
Target genes and oligonucleotide sequences for the forward and reverse primers (Integrated
DNA Technologies) are as follows-
β-actin
Forward: 5′-AGCCATGTACGTAGCCATCC-3′
Reverse: 5′-CTCTCAGCTGTGGTGGTGAA-3′
Cluster of differentiation factor 68 (CD68)
Forward: 5′-TTCTGCTGTGGAAATGCAAG-3′
Reverse: 5′-AGAGGGGCTGGTAGGTTGAT-3′
Interleukin-1β (IL1β)
Forward: 5′-GCAACTGTTCCTGAACTCAACT-3′
Reverse: 5′-ATCTTTTGGGGTCCGTCAACT-3′
(Moser et al. 2018)
Peroxisome proliferator-activated receptor-gamma coactivator (PGC 1α) (mouse)
Forward: 5′-GAAAGGGCCAAACAGAGAGA-3′
98
Reverse: 5′-GTAAATCACACGGCGCTCTT-3′
(Siddiqui et al. 2015)
IGF-1 ELISA- IGF-1 levels in serum from 8.5 to 9 month-old and 14.5 to 15 month-old ASO
and WT mice either in the control or FMD groups, was measured using a Mouse / Rat IGF-
I/IGF-1 Quantikine ELISA Kit (R&D Systems, MG100) and performed according to
manufacturer’s instructions.
Quantification and Statistical Analysis - GraphPad Prism v.8 was the software used for
statistical analysis and the figure legends described the statistical tests used, value of n for each
experimental group, and what n represents for each experiment that was conducted. All statistical
tests were two-sided and p-values <0.05 were noted as significant (*p<0.05, **p<0.01,
***p<0.001, ****p<0.0001). Differences between the means of two groups were tested with
unpaired 2-tailed student’s t-test comparison, and one-way or two-way ANOVA followed by
Tukey’s multiple comparison test. For the nest building behavior test, a non-parametric statical
test such as the Kruskal-Wallis was used to compare the different groups. Unless otherwise
specified in figure legends, all data are expressed as the mean ± SEM. All samples represent
biological replicates.
99
Chapter 5: Conclusion
5.1 Summary of findings and Significance
Previous research from our laboratory has shown that FMD cycles can ameliorate and / or
reverse symptoms in a number of different animal models for age-related, chronic inflammatory
diseases and that FMD cycles are safe and feasible for human subjects.
In chapter 2, we conclude that FMD cycles improve cognition in the 3xTg and E4FAD
mouse model of AD along with the reduction in amyloid beta, tau pathology and microglia
levels, increase in neurogenesis and reduction in NADPH Oxidase (Nox2) levels. In our long-
term study of FMD cycles, where male and female 3xTg mice were administered cycles of FMD
starting at 3.5 months of age for ~15 months (at ~18.5 months of age), we observed a reduction
in Aβ load and hyperphosphorylated tau in the hippocampus, a reduction in microglia levels
(CD68+, CD11b-ir cells), and modest shifts in the number of activated microglia.
In a E4FAD mouse model of AD, female mice were administered FMD at 3 months old
for 4 to 4.5 months. We observed that the E4FAD FMD mice had a higher spatial memory that
the E4FAD Control mice at the end of the study. This was accompanied by a reduction in Aβ
load in the hippocampus and cortex, increase in neurogenesis (as indicated by a higher number of
Ki67
+
and Sox2
+
cells) and a decrease in the levels of Nox2 in the cortex.
In our short-term study in 3xTg mice, where 5 cycles of FMD were given to 6.5-month-
old male and female 3xTg-AD mice till 8.5 months, we observed a reduction in IBA-1
expression in the hippocampus and an improvement in short-term memory in male 3xTg mice.
Here, the mice were euthanized after the 5
th
cycle of FMD before refeeding. A separate cohort of
male 3xTg mice in the short-term study were given 4 cycles of FMD and euthanized two days
post refeeding and female 3xTg were administered one cycle of FMD (no refeeding). It is
100
important to note the different results we obtained in Nox2 levels when the mice were euthanized
with respect to the FMD cycle. We found that Nox2 levels were significantly reduced in male
and female 3xTg after 5 cycles of FMD and also in female 3xTg mice after 1 cycle of FMD prior
to refeeding. Only in male 3xTg mice after 4 cycles of FMD and 2 days post refeeding, Nox2
levels were not significantly decreased. Thus, FMD can regulate Nox2 but the effect may not
continue during the refeeding phase. Results from 3xTg-AD/Nox2-KO and Apocynin treated
3xTg mice indicated that cognitive deficits, microglia levels and tau pathology to an extent,
could be affected through Nox2. Our findings from the different mouse models of AD are
summarized in Schematic 4.
Schematic 4. Summary of the effects of FMD on AD mouse models.
Graphical representation of the effects of FMD on the E4FAD and the 3xTg mouse models of Alzheimer’s disease, through
which it mediates AD-associated pathology, neuroinflammation, neural stem cells and Nox2 levels.
In chapter 4, we observed that FMD cycles for ~6.5 months improve motor co-ordination
in a genetic mouse of Parkinson’s disease that over expresses Human Alpha Synuclein. This
improvement was accompanied by a modest reduction in neuroinflammation (IL1β), microglial
AD Mice
Tau
Aβ
Neuronal
dysfunction/death and
cognitive impairment
FMD
Neuro-
inflammatory
proteins
Superoxide/
Peroxynitrite (O
2
-
/ONOO
-
)
Neural stem
cells
NOX2-KO
Apocynin
3xTg E4FAD
Microglia
Synaptic plasticity
Dysregulation ?
101
activation (CD68) and the size of Proteinase K resistant Alpha Synuclein aggregates. We will
add more mouse samples to the experiments to confirm our findings from our RT-qPCR analysis
and Proteinase K resistant Alpha Synuclein aggregates and will investigate the relationship
between Alpha Syn oligomers, aggregates and TLR2 Receptors in Microglia versus neurons,
along with NFkB translocation to the nucleus (microglia) and autophagy induction (neurons).
Schematic 5 summarizes the potential effects of FMD on the Alpha Synuclein Overexpressing
model.
Schematic 5. Summary of the potential effects of FMD on the ASO mouse model of PD.
Graphical representation of the potential effects of FMD in neurons and microglia in the ASO mouse model of Parkinson’s
disease. The results from the study reported here show that FMD cycles mediate Alpha Synuclein pathology, neuroinflammation,
microglial activation and improve motor co-ordination.
Through our findings we conclude that FMD cycles have a potential for slowing the
progression/treating neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease,
not just by ameliorating/reversing the symptoms of the disease but by targeting signaling
pathways related to neuroinflammation, microglial activation, oxidative stress and neurogenesis.
102
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Abstract (if available)
Abstract
A diet that mimics the effects of fasting without the adverse effects of water only fasting has been shown to prevent or slow the progression of different disease such as diabetes, cancer, multiple sclerosis and inflammatory bowel disease. Fasting Mimicking diet (FMD) also extended longevity in mice administered the diet starting at middle age and reduced disease risk factors for individuals from 20 to 70 years of age. Through the down regulation of Insulin-like Growth Factor 1 (IGF-1) and Protein kinase A (PKA) signaling, FMD was found to promote neurogenesis and enhance cognition as well as motor coordination in old mice. In a Multiple Sclerosis model, FMD reduced inflammatory cells as well as pro inflammatory cytokines.
We studied the effects of FMD cycles in genetic mouse models of Alzheimer’s and Parkinson’s disease. We found that FMD cycles improve cognition, ameliorate pathology, increase neurogenesis and reduce neuroinflammation the 3xTg and E4FAD mouse model of Alzheimer’s disease. FMD cycles also lowered oxidative stress marker, NADPH Oxidase (Nox2) levels in 3xTg mice. In a genetic mouse model of Parkinson’s disease in which human Alpha Synuclein is overexpressed, we found that long term cycles of FMD, improved moto co- ordination, reduced the size of proteinase K resistant Alpha Synuclein aggregates in the Substantia Nigra (SN) and reduced gene expression of neuroinflammation markers in the mid- brain.
The results from these studies indicate that FMD is a potential treatment option for neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease with its wide acting effects on neuroinflammation, neurogenesis, behavior (cognitive as well as motor) and ameliorating pathology in mouse models of these diseases.
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Creator
Lobo, Fleur Marie
(author)
Core Title
The effects of a fasting mimicking diet (FMD) on mouse models of Alzheimer's and Parkinson's disease
School
Leonard Davis School of Gerontology
Degree
Doctor of Philosophy
Degree Program
Biology of Aging
Degree Conferral Date
2022-08
Publication Date
07/26/2022
Defense Date
05/16/2022
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University of Southern California
(original),
University of Southern California. Libraries
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Alzheimer's disease,dietary interventions,FMD,OAI-PMH Harvest,Parkinson's disease
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English
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Electronically uploaded by the author
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Longo, Valter (
committee chair
), Andersen, Julie (
committee member
), Lee, David (
committee member
), Pike, Christian (
committee member
)
Creator Email
fleurlobo@gmail.com,flobo@usc.edu
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https://doi.org/10.25549/usctheses-oUC111375370
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etd-LoboFleurM-10995
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Lobo, Fleur Marie
Type
texts
Source
20220728-usctheses-batch-962
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Repository Email
cisadmin@lib.usc.edu
Tags
Alzheimer's disease
dietary interventions
FMD
Parkinson's disease