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Exploration and expansion of novel therapeutic strategies targeting brain metabolism in neurodegenerative diseases
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Exploration and expansion of novel therapeutic strategies targeting brain metabolism in neurodegenerative diseases
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Content
Exploration and expansion of novel therapeutic strategies targeting
brain metabolism in neurodegenerative diseases
By: Charles Clarke Caldwell
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Clinical and Experimental Therapeutics)
December 2018
2
Dedication:
I’d like to dedicate my doctoral thesis to my father, Charles H. Caldwell, who passed September
29
th
, 2018. He was always supportive and encouraged me through many challenging times. I
wish he was here to see this be completed. He will be loved and missed.
3
Acknowledgements
I would like to first acknowledge my mentor, Dr. Enrique Cadenas. He willingly took on me as
his responsibility when I changed labs and has given me his full support and guidance over the
last several years. He challenges and inspires me to be the best scientist I can be and to sit and
really think about science. I also must thank my two other committee members Dr. Giselle
Petzinger and Dr. Michael Jakowec for their guidance throughout the years and willingness to
help whenever needed. Thank you to Ron Irwin, Fei Yin, and Jia Yao for their teachings of
technical experimentation. Thank you to all my fellow lab members over the years including
Christine Solinsky, Lauren Klosinski, Manuil Desai, Ishan Patil, and Karren Wong. Thank you to
Alicia Warnecke and Elliott Cheung for your integral assistance on the HD exercise project.
I must stand the entire regulatory science department especially Michael Jamieson who always
helped in expanding my industry knowledge. I must also thank Ed Lieskovan for his class on
pharma commercialization and Professor Steven Mednick at USC Marshall for my work in the
business school.
Lastly, I thank my family for being there by my side through all the ups and downs during the
past 5 years and before those. My mother, the strongest person I know, and to my father someone
who was so full of life it was almost impossible to imagine it without him. And my brother who I
will always stand beside as I’m sure he will need me just as I’ve needed him. To my
grandparents thank you for your support and inspiration to challenge myself academically,
physically, and emotionally.
4
TABLE OF CONTENTS
Title Page……………………………………………………………………………………….…1
Dedication………………………………………………………………………………………....2
Acknowledgements………………………………………………………………………………..3
Table of Contents……...…………………………………………………………………………..4
Abstract……………………………………………………………………………………………5
Chapter 1: Introduction……………………………………………………………………………6
Chapter 2: Sex Differences in Brain Metabolism and Therapeutic Response in a 3xTgAD Mouse
Model……………………………………………………………………..……………………...23
Chapter 3: Applications for impacting Parkinson’s through exercise, clinical log……….……..56
Chapter 4: Exercise Rescues Mitochondrial Function and Motor Behavior in a CAG140 KI
Advanced Huntington’s Disease Mouse Model…………………………………………………64
Chapter 5: Concluding Remarks…………………………………………………………....……84
References………………………………………………………………………………………..87
5
Abstract
This thesis is a collection of work by Charles C. Caldwell during his doctoral studies at
University of Southern California School of Pharmacy in the major of Clinical and experimental
therapeutics. Topics of focus include brain aging and neurodegenerative disease, sex differences
in animal models and therapeutic response, clinical outcomes in comparison with preclinical
investigations, and exercise as a viable modulator of brain energy metabolism.
Neurodegenerative diseases in this thesis include Alzheimer’s, Parkinson’s, and Huntington’s.
Drug and non-drug-based interventions impacted glycolytic and mitochondrial metabolic
function resulting in cognitive and motor functional improvements.
6
CHAPTER 1
INTRODUCTION
Neurodegenerative diseases affect millions of people in the US today 5 million
Alzheimer's disease (AD) (5 million); Parkinson's (PD) (1 million); multiple sclerosis (MS)
(400,000); amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) (30,000), and
Huntington's disease (HD) (30,000).In 30 years at the current growth rate 12 million Americans
will suffer from some form of neurodegenerative disease (Alzheimer's, 2016; Janca, 2004;
Spencer et al., 2016). Several of these disorders have similar phenotypic profiles of cognitive
dysfunction including Alzheimer’s, Parkinson’s, and Huntington’s. Current treatment options
from drug development have only come up with mild symptomatic relief, but not current therapy
prevents, perturbs, or reverses these cognitive deficits. Current drugs for Alzheimer’s have
focused on the acetylcholine and glutamate pathways as well as the removal of amyloid plaque.
However, more mechanistic work has shown that the underlying problem likely stems from a
metabolic dysfunction of the brain long before plaque formation. Huntington’s disease also
seems to be influenced by a metabolic component. The disease progression is heavily dependent
on the mutation of the huntingtin gene and the number of CAG repeats. However, influencing
brain metabolism may hold a key part in disease treatment. Parkinson’s disease is a
neurodisorder involving dopamine utilization. Many current symptomatic treatments impact
dopamine pathways. Parkinson’s disease progression is also influenced by brain metabolic
dysfunction (Alexi et al., 2000; Berman and Hastings, 1999; Bogdanov et al., 2008; Chaturvedi
and Beal, 2013; Keeney et al., 2006). Brain metabolic research in these neurodegenerative
diseases have investigated glucose uptake, mitochondrial function, and insulin resistance as
possible avenues of intervention (Bingham et al., 2002; Gibson et al., 1998; Hauptmann et al.,
2009b; Jiang et al., 2013; Mosconi, 2005; Sancheti et al., 2014; Yin et al., 2016a; Yin et al.,
2016b).
7
Aging and metabolic control
Aging can be broadly described as time-dependent impaired function ultimately leading
to death. However, many factors influence the rate of this process and several of these factors are
modifiable. Aging is the primary risk factor for the majority of human morbidities including
cardiovascular disorders, diabetes, cancers, and neurodegenerative diseases(Lopez-Otin et al.,
2013). Some biochemical hallmarks leading to these age-related morbidities include genomic
instability, telomere attrition, epigenetic alterations, cellular senescence, altered intercellular
communication, and mitochondrial dysfunction.
It is suggested that these aging hallmarks are under metabolic control which hold the key
to extending lifespan; genomic instability leads to decreased glucose metabolism and mitophagy
hyperpolarization, telomere attrition leads to decreased mitochondrial biogenesis, cellular
senescence leads to decreased NAD
+
availability (Lopez-Otin et al., 2016). Many factors are
attributed to aging and age related neurodegenerative disease including environmental toxicants,
high caloric and fat diets, lack of proper nutrient supplementation, and sedentary lifestyle. Also
referred to as the Westernized lifestyle, this combination of high caloric intake and extended
sedentariness is likely the main contributor to the steady rise in neurogenerative disease
incidence in the US. This western diet also exposes people to environmental toxicants such as
certain pesticides and fertilizers(Lopez-Otin et al., 2016).
Currently there are very few effective interventions to combat age associated disease. A
well-studied and accepted effective early intervention of accelerated aging is caloric restriction
which may include specific dietary changes (Finkel, 2015; Longo et al., 2015; Madeo et al.,
2015). Another is cardiovascular and muscular exercise; previous work has shown how exercise
can increase neurotransmitters such as glutamate and aspartate as well as normalize low NAD
+
levels. Exercise has also been linked to increased acetyl-CoA pools and improved metabolism in
models of high-fat diet (Clark-Matott et al., 2015; Finkel, 2015). However, specific mechanisms
of how exercise directly impacts brain metabolism in neurodegenerative disease requires further
research.
8
Alzheimer’s Disease
Since 1998, there have been 101 failed Alzheimer’s trials (Thies and Bleiler, 2013).
Currently available drugs offer moderate symptom alleviation (Thies and Bleiler, 2013). No
therapeutic strategies have demonstrated clinically significant disease modifying benefits to halt or
reverse cognitive decline.
Most of the therapeutic candidates have focused on reduction or reversal of AD pathology
based on the amyloid plaque hypothesis. Several anti-amyloid drug candidates have failed in late-
stage clinical trials (Feldman et al., 2014; Karran et al., 2011). Despite preclinical success in cell
lines and animal models, most therapeutic candidates for AD failed to show any significant effect on
cognitive function at clinical stages (Thies and Bleiler, 2013). These failures can be attributed to
multiple factors that arise during drug development in both preclinical and clinical settings.
As multifactorial diseases present differently, responses to therapies also differ. For example,
unhealthy diet and exercise may have different impact on individuals and require different treatment
strategies than those individuals predisposed to genetically inherited familial diseases (Morris et al.,
2014). Gender, genetic risks, and age are important variables that should be considered during the
development stage for AD therapeutics (Brinton, 2008a; Irwin, 2014). As well, the dosing regimen,
formulation, and the route of administration all have significant effects on clinical success (Irwin,
2014). Past approaches targeting moderate and severe AD pathology have had minimal success in
part because of the single target strategy for a multifactorial pathology. In contrast, targeting the
affected biological systems at specific stages of disease progression may have greater likelihood of
success in non-familial AD.
The presymptomatic and prodromal stages of AD are within a window of opportunity to have
the greatest impact on lowering the risk and incidence of AD (Figure 1). Targeting brain metabolism
and mitochondrial function are relevant to the hypometabolism and impaired mitochondrial
bioenergetics that are among the earliest pathogenic events.
9
Fig. 1 The five stages of Alzheimer’s disease pathology and three therapeutic treatment windows.
The prodromal state encompasses pre-symptomatic and mild cognitive impairment stages of AD.
White line: progression of cognitive decline through the five stages of AD (De Santi et al., 2001;
Gibson and Shi, 2010a; Hauptmann et al., 2009a; Ishii et al., 1997; Mosconi et al., 2008; Nicholson
et al., 2010; Protas et al., 2013).
Parkinson’s Disease
Parkinson’s disease (PD) is a slow, progressive neurological disorder characterized by a large
number of motor and non-motor features (Fau, Tolun, & Toru, 2009). However, currently there is no
cure for the disease (Kernohan & Waldron, 2011). Since 1962, Doshay relayed the benefits of
10
physical therapy on patients with PD. In addition, several studies have addressed the beneficial effect
of exercise on the functional outcomes in patients with PD including: posture, gait, balance, activities
of daily living, and muscle strength (Rodrigues-de-Paula, Teixeira-Salmella, & Cardoso, 2011;
Nallegowda, Singh, Handa, Khanna, Wadhwa, Yadav, et al., 2004; Allen, Sherrington, Paul, &
Canning, 2011; Dibble, Addison, & Papa, 2009).
Functional brain activation during the execution of acute motor tasks and motor learning of hand and
finger tasks mainly from studies involving healthy subjects (Tashiro et al. 2008, Fukuyama et al.
1997, Doyon et al. 2009), substantially less is known about the functional reorganization of the brain
after long-term aerobic exercise, particularly in PD patients (Toma et al. 2002). Most studies on
extensive motor training in human subjects have focused on the learning of finger tasks during an
fMRI imaging session (Rowe et al. 2012, Munte et al. 2002), with a near absence of studies
examining the effects of long-term ET on functional brain changes (Alberts et al. 2011, Beall et al.
2013). An improved understanding of the effects of long-term aerobic training on brain functional
activation and NOS impact, in PD patients, would be helpful for directing targeted
neurorehabilitative strategies, and in understanding motor compensations in PD.
Huntington’s Disease
HD is characterized by progressive decline in cognitive and motor functions with
neuropsychiatric disturbances leading ultimately to premature death. The major pathological
findings include severe degeneration of striatal medium spiny neurons (MSNs) and the cerebral
cortex, particularly the prefrontal and frontal cortex. In the striatum, there is a preferential loss of
the dopamine D2 receptor (DA-D2R)-containing MSNs that mediate the indirect pathway
compared to direct pathway dopamine D1 receptor (DA-D1R)-containing MSNs. Defects in the
respiratory chain in HD have been observed in early biochemical studies. Severe reduction in the
activity of complex II/III and milder reduction of complex IV were found in post mortem
samples of the caudate/putamen in HD patients. No changes were observed in pre-symptomatic
patients. The cerebral cortex showed minor changes in respiratory chain enzymes. Reduced
11
activity of other enzymes of oxidative metabolism in the striatum was also reported. Massive
loss of aconitase activity has been found in the caudate (~90%), and putamen (~70%). Some of
these biochemical markers can help target future therapies.
Current strategies targeting mitochondria and bioenergetics in neurodegenerative disease
The integrity and viability of the bioenergetic system is a fundamental determinant of
synaptic and brain function (Brinton, 2008a; Ding et al., 2013a; Swerdlow, 2014; Swerdlow et al.,
2014). Although the human brain accounts for 2% of the body’s mass, it consumes 20% of the
body’s fuel supply for adenosine triphosphate (ATP) production (Brinton, 2008a). The bioenergetic
system consists of obligatory processes that are tightly coupled, including substrate supply,
transporters, and the catalytic machineries required for oxidative phosphorylation and ATP
generation (Figure 2). Compromised brain metabolism is an early indicator of Alzheimer’s disease in
both preclinical and clinical investigations (Ding et al., 2013a). Previous studies have suggested that
a decrease in brain bioenergetics may be a useful biomarker to predict disease decades before
symptoms (Brinton, 2008b; Cardoso et al., 2004b; Swerdlow, 2007; Swerdlow and Khan, 2009).
Decreases in mitochondrial bioenergetics, metabolic enzyme expression and activity, cerebral
glucose metabolism, along with increased oxidative stress, Aβ deposits within mitochondria, and
expression of Aβ-binding alcohol dehydrogenase (ABAD) are associated with the prodromal state of
AD (Blass et al., 2000; Cardoso et al., 2004a; Cardoso et al., 2004b; Gibson et al., 1988; Parker,
1991; Perry et al., 1980; Sorbi et al., 1983).
12
Fig. 2 Dysfunction of the brain energy production system precedes Alzheimer’s disease
pathology and neuronal death. Strategies to prevent mitochondrial dysfunction include
multiple points of therapeutic in tervention: 1) Glucose or alternative fuel for substrate
supply; 2) glycolysis; 3) TCA cycle and electron transport chain (ETC); 4) oxidative
stress; 5) apoptosis. Mitochondrial impairments produce free radicals causing oxidative
damage; ROS and caspase proteases can block the neuroprotective mitophagy pathway.
Mitochondrial dysfunction and endoplasmic reticulum (ER) stress activate apoptotic
pathways that lead to neuronal loss and continuation of the AD pathology spectrum.
Glucose uptake and substrate supply
Decreased glucose metabolism is an early hallmark of the prodromal AD stage (Reiman et
al., 2012). Brain hypometabolism and deficits in mitochondrial bioenergetics have long been
documented in both preclinical and clinical AD research. Decrements observed in cerebral glucose
metabolism using fluoro-2-deoxyglucose positron emission tomography (FDG-PET) and brain
13
volume using magnetic resonance imaging (MRI) are early signs of bioenergetic decline in the
prodromal state of AD (Protas et al., 2013) (Figure 1). Observations from a clinical trial of the
Dominantly Inherited Alzheimer’s Network (DIAN) suggested several surrogate disease markers,
including compromised FDG-PET signal in specific brain regions (posterior cingulate cortex and
prefrontal cortex) vulnerable to development of AD pathology, that arise in AD patients decades
before cognitive symptoms (Carrillo et al., 2013; De Santi et al., 2001; Gibson and Shi, 2010a;
Hauptmann et al., 2009a; Ishii et al., 1997; Mosconi et al., 2008; Nicholson et al., 2010; Yao et al.,
2009).
At the substrate level, glucose transport across the blood brain barrier into neurons and glial
cells require glucose transporters glial GLUT1 (55 kD and 45 kD), neuronal GLUT3, and insulin-
dependent GLUT4 (Ding et al., 2013a). Glycolysis, TCA cycle, and mitochondrial OXPHOS are
then coordinated to generate ATP (Ding et al., 2013a; Swerdlow, 2014). Compromised glucose
uptake and metabolism provide a therapeutic target for AD prevention and intervention. Therapeutic
candidates that target glucose metabolism could address the hypometabolic phenotype. If glucose
hypometabolism in brain is a causative factor in development of AD, then detection, prevention and
reversal of bioenergetic decline represent a therapeutic target window for AD (Chetelat et al., 2003).
Insulin is a therapeutic candidate to promote glucose metabolism in the brain (Cholerton et al., 2013;
Watson and Craft, 2003) (Table 1). Insulin plays an essential role in energy metabolism in the brain
with receptors densely populating the medial temporal regions of the brain required for memory
formation (Watson and Craft, 2003). Additionally insulin-sensitive glucose transporters (GLUT4) are
expressed in regions supporting memory and cognitive function (Watson and Craft, 2003). Insulin-
resistance, which is the reduced sensitivity of insulin in targeted tissues important for cognitive
function, increases risk of dementia (Cholerton et al., 2013). Impaired insulin responsiveness and
dysfunctional glucose utilization have been documented in post-mortem Alzheimer’s brains (de la
Monte, 2014a, b). Intranasal insulin was tested in a randomized, double blind, placebo controlled
clinical study of 64 participants with MCI and 40 participants with mild to moderate AD. Insulin
induced modest recovery of memory function and preservation of glucose uptake (Craft et al., 2012;
14
Reger et al., 2008). A larger scale phase II/III trial is currently underway to examine the effects of
intranasally-administered insulin on cognition, entorhinal cortex and hippocampal atrophy, and
cerebrospinal fluid (CSF) biomarkers in amnestic mild cognitive impairment (aMCI) or mild AD
(ClinicalTrials.gov identifier: NCT01767909).
In addition to glucose, an alternative fuel source, ketone bodies, is used for cellular processes
when glucose and carbohydrate supply is low (Brinton, 2008b; Panov et al., 2014) (Table 1) (Figure
2). Ketone bodies are formed in the liver from fatty acid oxidation and are transported to the brain
(Ding et al., 2013a; Magistretti, 2006; Panov et al., 2014). Ketone bodies, transported into the cell via
monocarboxylate transporters (MCTs), bypassing glycolysis, are subsequently utilized by a series of
ketolytic enzymes such as succinyl-CoA:3-ketoacid CoA transferase (SCOT) instead of pyruvate
dehydrogenase (PDH) to produce Acetyl-CoA, which condenses with oxaloacetate and enters the
TCA cycle for energy production (Ding et al., 2013a; Panov et al., 2014). Several therapeutic
strategies aim to enhance brain bioenergetics through supplementation of ketone bodies, including
acetoacetate and β-hydroxybutyrate (Owen et al., 1967; Swerdlow, 2011) or dietary induction of
ketogenesis (Kashiwaya et al., 2013). However, patient compliance on ketogenic diets is challenging
due to its high fat and low carbohydrate content. In addition, there are studies indicating that ketone
bodies have no effect on AD pathology despite benefits on motor performance (Beckett et al., 2013;
Brownlow et al., 2013). Future research could develop forms of ketogenic supplementation for
alternative energy.
Mitochondrial bioenergetics
Multiple experimental paradigms, ranging from in vitro cell model systems and genomic
analyses in animal models to postmortem autopsy of human brain and human brain imaging indicate
deficits in mitochondrial function are consistent antecedents to AD development (Gibson and Shi,
2010a; Hauptmann et al., 2009a; Nicholson et al., 2010; Yao et al., 2009). A decline in
mitochondrial function can occur decades prior to clinical diagnosis of AD and thus may serve as a
biomarker of AD risk as well as a therapeutic target (Hauptmann et al., 2009a; Swerdlow, 2009,
15
2014; Yao et al., 2009). Preclinical in vitro and in vivo AD models have demonstrated a decline in
mitochondrial function prior to AD pathology including reduced mitochondrial respiration, decreased
metabolic enzyme expression and activity, increased oxidative stress, and increased mitochondrial
Aβ load and ABAD expression (Cardoso et al., 2004a; Cardoso et al., 2004b; Hauptmann et al.,
2009a; Mosconi et al., 2008; Nicholson et al., 2010; Yao et al., 2009). A series of mitochondrial
enhancer candidates have been proposed and investigated in preclinical and clinical studies for AD
prevention and treatment (Table 2).
Multiple candidate molecules target the electron transport chain (ETC). Coenzyme Q (CoQ)
and its synthetic water-soluble analogue, idebenone, have been proposed for AD treatment (Dumont
and Beal, 2011; Mancuso et al., 2010; Swerdlow, 2014). CoQ is imbedded in the mitochondrial inner
membrane and transports electrons from complex I/II to complex III in the ETC. In addition, CoQ
can function as a reactive oxygen species (ROS) scavenger (Mancuso et al., 2010). While CoQ
supplementation has benefit in persons with CoQ synthesis disorders and in preclinical mouse
models of AD, it is ineffective as a therapeutic in persons with AD (Dumont and Beal, 2011;
Swerdlow, 2011, 2014). In a randomized, double-blind, multicenter study with 450 mild to moderate
AD participants, Idebenone showed minimal cognitive benefit (Bergamasco et al., 1994; Gutzmann
and Hadler, 1998; Weyer et al., 1997) but was not approved for treatment of AD based on results not
reaching statistical significance in larger trials (Gutzmann and Hadler, 1998; Thal et al., 2003; Weyer
et al., 1997). Methylene blue can enhance cytochrome c oxidase (COX) activity through direct
electron donation (Atamna et al., 2008; Callaway et al., 2004). In a preclinical AD mouse model,
methylene blue treatment reduced Tau-neurofibrillary tangle burden (Atamna and Kumar, 2010).
Clinical investigations of methylene blue as a treatment for AD have not been conducted. Menadione
and ascorbate together can act as complex IV substrates and sustain mitochondrial ETC respiration
when complex III is compromised (Eleff et al., 1984). Other compounds that enhance mitochondrial
ETC and oxidative phosphorylation include nicotinamide, a precursor to the complex I substrate,
NADH, and riboflavin, a precursor to the complex II substrate, FADH2 (Schoenen et al., 1998).
While outcomes of research on these molecules have shown promising potential in preclinical
16
studies, their efficacy clinically is unlikely to be substantial as they target specific components of the
bioenergetic system instead of the entire system. A case in point is creatine, which is proposed to
increase energy storage capacity and could be used to generate ATP under high-energy demands.
However, creatine failed in clinical trials and in some cases even caused negative effects (Shefner et
al., 2004; Verbessem et al., 2003).
Activation of the peroxisome proliferator-activated receptor-gamma (PPAR-ɣ) and the
peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) pathway promote
mitochondrial biogenesis (Dumont and Beal, 2011; Wenz et al., 2008; Wenz et al., 2011).
Therapeutic candidates in this class include PPAR-ɣ agonist thiazolidinediones, pioglitazone and
rosiglitazone, as well as the PGC1α activator Bezafibrate. Activation of PGC1α by Bezafibrate was
reported to improve cell bioenergetics and decrease mitochondrial dysfunction in cell culture and
animal models (Wenz et al., 2008; Wenz et al., 2011).
Preclinically, pioglitazone restored cerebrovascular function, reduced oxidative stress, and
increased mitochondrial respiration (Escribano et al., 2009; Nicolakakis et al., 2008; Strum et al.,
2007). Rosiglitazone stimulated neuronal mitochondrial biogenesis and reduced memory deficits in
mouse models of AD (Escribano et al., 2009; Nicolakakis et al., 2008; Strum et al., 2007). A small
clinical trial of 32 mild AD patients showed significant improvements with pioglitazone treatment on
both the AD Assessment Scale-Cognitive subscale scores and Wechsler Memory Scale-Revised
Logical Memory Performance tests (Hanyu et al., 2009; Sato et al., 2011). The patients also had
improved cerebral blood flow in parietal lobes (Sato et al., 2011). A phase III clinical trial of
pioglitazone for mild cognitive impairment due to Alzheimer’s disease is currently underway
(ClinicalTrials.gov identifier: NCT01931566). Rosiglitazone tested in MCI and early AD showed
improved delayed recall (Watson et al., 2005), but failed to show significant cognitive benefits in a
subsequent larger trial with over 1400 mild to moderate AD patients (Risner et al., 2006)
(ClinicalTrials.gov identifier: NCT00490568). One major challenge for these candidates is they have
poor blood brain barrier (BBB) penetration (Ghosh et al., 2007). However, if co-transported through
17
the BBB, rosiglitazone and other thiazolidinediones could be therapeutically beneficial in preventing
AD.
Another potential target of the mitochondrial biogenesis pathway is the mitochondrial
transcription factor A (TFAM), which is involved mitochondrial biogenesis, mtDNA replication,
transcription, and removal of homoplasmic mtDNA mutations (Khan and Bennett, 2004). In vitro
administration of TFAM reportedly increased mitochondrial respiration rates, biogenesis, and
mtDNA levels (Ekstrand et al., 2004; Kang et al., 2007; Scarpulla, 2008). The potential of TFAM as
a therapeutic is unlikely due to its large size and difficulty for BBB penetration (Pardridge, 2005).
However, targeting expression of TFAM could be a therapeutic strategy to enhance mitochondrial
bioenergetics.
Mitochondrial enhancers have been demonstrated to be effective in preclinical models of AD
whereas there are few clinical trials that have tested this strategy directly. Targeting mitochondrial
directly assumes that the bioenergetic system of substrate transporters and metabolism are fully
functional. This is unlikely to be the case in the prodromal and later stages of the disease based on
clinical FDG-PET data indicating impaired glucose metabolism decades prior to symptom
development. Mitochondrial function is inextricably linked to the upstream substrate supply and
metabolism. Thus, increasing mitochondrial function in the presence of dysfunctional substrate
transport and metabolism could exacerbate degeneration. However, therapeutic strategies that
promote each functional domain of the bioenergetic system including mitochondrial function could
have benefit.
Oxidative damage as a therapeutic target
A well-documented indicator of compromised mitochondrial function is oxidative stress
(Cadenas, 2004; Dumont and Beal, 2011; Dumont et al., 2010a; Lin and Beal, 2006; Yap et al.,
2009). Oxidative stress is primarily caused by excessive ROS produced by impaired electron
transport, ER stress, and peroxisomes (Dumont and Beal, 2011; Kubli and Gustafsson, 2012; Lin and
Beal, 2006; Wang et al., 2014; Yao et al., 2009) (Figure 2). Decreases in enzymatic antioxidant
18
defense capacity including multiple superoxide dismutases (SOD), peroxiredoxins and glutathione
(Beal, 2005; Reddy and Beal, 2008), further exacerbates oxidative damage (Dumont and Beal, 2011;
Dumont et al., 2010a; Lin and Beal, 2006; Yap et al., 2009). Oxidative damage of multiple cellular
components has been documented in both preclinical models of AD and in persons with the disease
(Butterfield et al., 2014; Wang et al., 2014). Key enzymes involved in mitochondrial function, such
as pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (αKGDH), are often targeted
by ROS, leading to deceased enzyme activity and decreased efficiency of mitochondrial electron
transport (Starkov et al., 2004) (Figure 2). In AD elevated oxidative stress is detectable in the form of
lipid peroxides, 8-oxoguanine, and other oxidized proteins (Nunomura et al., 2004; Reddy, 2006;
Zhang et al., 2007; Zhao and Brinton, 2007). In parallel, oxidative stress have been demonstrated to
increase A production in vitro and in vivo (Wang et al., 2014; Zhang et al., 2007).
Several candidates have been proposed to prevent and/or reduce oxidative damage and have
been investigated as treatments for AD (Table 3). Deficits in plasma levels of antioxidants are well
documented in AD patients (Lopes da Silva et al., 2014; Mi et al., 2013; Shigenaga et al., 1994).
Early research administering antioxidants for AD treatment focused on vitamin C and vitamin E.
These two vitamins are significantly reduced in the plasma levels of AD patients (Lopes da Silva et
al., 2014). Multiple preclinical studies using transgenic AD mouse models of vitamin C and vitamin
E indicated decreased lipid peroxidation, memory deficits, and beta amyloid plaque burden
(Bjelakovic et al., 2007; Harrison et al., 2009b; Sung et al., 2004). However, clinically neither
vitamin C nor vitamin E showed significant benefits for cognitive function or delay of AD
progression (Jack et al., 2008; Parnetti et al., 1995; Shoulson, 1998). Some studies indicated negative
effects of high dose vitamin E on cognitive function and increased risk for mortality (Miller et al.,
2005). In addition to vitamin C and vitamin E, several other antioxidant vitamins have been
investigated due to their deficient levels in AD patients, including vitamin A, vitamin B 12, and
vitamin D (Lopes da Silva et al., 2014; Shah, 2013). These vitamins failed to show significant
efficacy when used in clinical settings (ClinicalTrials.gov identifier: NCT00235716), despite positive
19
outcomes in preclinical models. Currently, these vitamins are often used in combination with other
therapeutics based on their general health benefits.
Micronutrients and minerals have been investigated as potential therapeutics for AD. These
include, but are not limited to, flavonoids (such as quercetin, morin, or baicalein), β-carotene,
curcumin, zinc, folic acid, and selenium. Most of these showed little promise as an AD therapeutic
on their own, but when combined together in formulations they improved cognitive function in
transgenic AD mice and reduced oxidative stress (Grodstein et al., 2003; Morris et al., 2002).
Other antioxidant compounds include curcumin, which has been demonstrated to induce
multiple benefits in AD mice. In addition to its strong antioxidant ability, it has anti-inflammatory
activity, reduces amyloid plaque burden, and partially restored distorted neuritis (Garcia-Alloza et
al., 2007; Lim et al., 2001; Yang et al., 2005; Zhang et al., 2006). A current Phase II clinical trial that
combines curcumin and yoga therapy aims to treat MCI (ClinicalTrials.gov identifier:
NCT01811381). Resveratrol found in red grape skin is a potent antioxidant that has been shown to
reduce amyloid plaque burden and improve memory deficits in transgenic AD mice
(Karuppagounder et al., 2009; Vingtdeux et al., 2010). Resveratrol activates the AMPK pathway, and
stimulates activity of NAD-dependent deacetylase sirtuin-1 (SIRT1) (Beher et al., 2009; Canto et al.,
2009), which subsequently activates the PGC1a metabolic regulatory pathway and promotes
mitochondrial biogenesis (Baur et al., 2006; Canto et al., 2010; Timmers et al., 2011; Um et al.,
2010). Several clinical trials are currently underway to investigate the efficacy of resveratrol
including a Phase II on mild to moderate AD (ClinicalTrials.gov identifier: NCT01504854), a Phase
III on mild to moderate AD (ClinicalTrials.gov identifier: NCT00743743), and a Phase IV on MCI
which combines resveratrol with omega-3 (ClinicalTrials.gov identifier: NCT01219244).
There is also a set of compounds including melatonin that promote expression of
mitochondrial antioxidant enzymes such as superoxide dismutase (SOD) or glutathione (Feng et al.,
2006; Hardeland, 2005; Larson et al., 2006; Olcese et al., 2009). Mitoquinone mesylate (MitoQ) has
been proposed for treatment of AD and other neurodegenerative diseases because its antioxidant
activity localizes to the mitochondrial inner membrane to prevent oxidative damage (McManus et al.,
20
2011). MitoQ has shown bioenergetic benefits in AD mouse models, but the clinical benefit of
MitoQ for AD patients is yet to be determined (McManus et al., 2011).
Overall strategies based on antioxidants and micronutrients have shown promise in
transgenic AD models, but their clinical therapeutic efficacy has not been established under disease
conditions. However, their therapeutic potential in combination with factors that target the entire
bioenergetic system in the at risk and prodromal stages of the disease has yet to be investigated
(Figure 1).
Apoptosis and mitophagy as therapeutic targets
Prolonged deficits in bioenergetics together with elevated oxidative stress lead to activation
of apoptotic pathways, impaired mitophagy, and ultimately neuronal death (Gottlieb and Carreira,
2010). Mitochondrial autophagy, often referred to as mitophagy, is a highly dynamic process for
disposal and recycling of unhealthy mitochondria (Gottlieb and Carreira, 2010). A balance between
mitophagy and mitochondrial biogenesis provides an efficient energy transducing system required for
neuronal survival (Vosler et al., 2009) whereas mitochondrial dysfunction contributes to neuronal
death and AD pathology (Figure 2) (Bachurin et al., 2003; Feng et al., 2006; Lin and Beal, 2006).
Elevated oxidative stress and induction of apoptotic proteases can inactivate mitophagy and impair
pathways required for clearance of aberrant mitochondria (Andreux et al., 2013; Chen and Chan,
2009; Dumont and Beal, 2011; Dumont et al., 2010a).(Kubli and Gustafsson, 2012).
Several therapeutic strategies target the autophagy and mitophagy pathways (Table 4).
Rapamycin, also known as sirolimus, is being investigated for AD based on its potential to induce
autophagy in several neurodegenerative disease models (Harrison et al., 2009a; Jung et al., 2010;
Kapahi et al., 2010). Mechanisms of rapamycin action include removal of damaged mitochondria and
cells with dysfunctional mitochondria via mTOR-dependent activation of autophagy (Jung et al.,
2010; Kapahi et al., 2010). Rapamycin extends life span in aged mice (Harrison et al., 2009a). In
transgenic AD mice, rapamycin reduced amyloid plaque and prevented cognitive decline (Harrison et
al., 2009a; Jung et al., 2010; Spilman et al., 2010). However, the therapeutic development of
21
rapamycin for treatment of neurodegenerative diseases was largely hampered due to severe side
effects including lung toxicity, diabetes, and cancer (Spilman et al., 2010).
Latrepirdine, also known as Dimebon, is an antihistamine that in cell culture reduced
swelling of mitochondria under Aβ stress and stabilized mitochondrial membrane potentials
(Bachurin et al., 2003; Zhang et al., 2010). Latrepirdine is proposed to interact with glutamate
receptors, block voltage-dependent calcium channels, and inhibit mitochondrial permeability and
thereby suppressed unnecessary mitophagy or apoptosis. (Grigorev et al., 2003; Lermontova et al.,
2001). In a phase II clinical trial in moderate AD patients, it significantly improved cognitive
function over placebo (Doody et al., 2008). However, these results were not confirmed
(Bezprozvanny, 2010) as Latrepirdine/Dimebon later failed in a larger phase III trial on moderate to
severe AD patients (Miller, 2010) (ClinicalTrials.gov identifier: NCT00912288).
Nuclear response factor 2 (Nrf2) and Nrf2/ARE have been proposed as a therapeutic target
for autophagy and mitophagy. In transgenic AD mice intrahippocampal injections of the lentiviral
vector expressing Nrf2 decreased amyloid plaque, reduced learning deficits, and protected against Aβ
induced cell death. (Kanninen et al., 2009; Kanninen et al., 2008). Synthetic triterpenoids have been
demonstrated to induce expression of Nrf2 and to protect against cell death in both in vitro and in
vivo experiments (Liby et al., 2005; Liby et al., 2007). Development of strategies that target this
Nrf2/ARE pathway are at an early stage and require substantial translational research.
Preclinical strategies targeting autophagy and mitophagy pathways in AD models have
shown cognitive benefits cognitive benefits. Substantial preclinical discovery and translational
research remain to be conducted to advance this therapeutic target.
Exercise as an intervention for neurodegenerative disease progression
Exercise has been studied and shown promise as a possible intervention strategy in
several cognitive diseases including Alzheimer’s, Parkinson’s, and Huntington’s (Fritz et al.,
2017; Machado et al., 2017; Oliveira de Carvalho et al., 2018; Petzinger et al., 2011). Though
there are difficulties with controls, patient compliance, and tailoring exercise regimens to
22
specific patient health profiles exercise interventions for neurodegenerative diseases are being
utilized in numerous clinical trials (NCT02000583; NCT02074215; NCT02195596;
NCT00591344; NCT02267785 NCT03344601; NCT01879267). The success of these
interventions in trial and in general clinical settings warrants continued research into the
mechanisms behind the functional benefits of exercise in the cognitively impaired. Parkinson’s
and exercise expert Dr. Giselle Petzinger explains several mechanisms for which exercise
influences the Parkinson’s brain. Exercise enhances cognition and motor behavior in Parkinson’s
through facilitating dopamine neurotransmission and synaptogenesis, attenuating dopamine
depletion and glutamatergic drive (Kintz et al., 2017; Petzinger et al., 2011; Petzinger et al.,
2007; Stefanko et al., 2017; Wang et al., 2016 ). An expansion of this work should investigate
the likely metabolic change in glucose utilization, insulin resistance, and mitochondrial
respiratory function. Previous exercise research in Huntington’s disease models has focused on
behavioral outcomes due to exercise and changes in circuitry and blood flow (Stefanko et al.,
2017). The metabolic changes due to exercise induced elevated nitric oxide concertation in
Huntington’s brain is discussed in Chapter 4.
Disclaimer
A significant portion of the content of Chapter I and portions of the introduction of
Chapter II were taken directly from my previous published work during my
doctoral studies.
Caldwell, C.C., Yao, J., and Brinton, R.D. (2015). Targeting the prodromal stage
of Alzheimer's disease: bioenergetic and mitochondrial opportunities.
Neurotherapeutics 12, 66-80.
23
CHAPTER 2
SEX DIFFERENCES IN BRAIN METABOLISM AND THERAPEUTIC RESPONSE IN A
3XTGAD MOUSE MODEL
Abstract
Alzheimer’s disease (AD) has a complex and progressive neurodegenerative phenotype, with
hypometabolism and impaired mitochondrial bioenergetics among the earliest pathogenic events.
Bioenergetic deficits are well documented in preclinical models of mammalian aging and AD,
emerge early in the prodromal phase of AD, and in those at risk for AD.
Understanding the bioenergetic adaptations that occur during aging and AD led us to focus on a
systems biology approach that targets the bioenergetic system rather than a single component of
this system. Bioenergetic system-level therapeutics personalized to bioenergetic phenotype
would target bioenergetic deficits across the prodromal and clinical stages to prevent and delay
progression of AD. We introduce a novel therapeutic formulation that acts on the multifaceted
bioenergetic system to help reduce cognitive and brain metabolic decline in the 3xTgAD mouse
model.
Additionally, little is understood regarding the possible differences between genders amongst the
same phenotype in the 3xTgAD model. This study hopes to illuminate some of the characteristic
variances between the two genders in baseline and in response to treatment.
We aim to develop a novel nutraceutical formulation that contains non-feminizing estrogenic
compounds and mitochondrial enhancer substrates to promote brain metabolic activity. We
predict this unique combination will counterbalance the bioenergetic system decline in the
3xTgAD Alzheimer’s mouse model. We hypothesize that a mechanistic approach that targets the
system of bioenergetics in brain to promote glucose transport, glycolysis, and mitochondrial
respiration will prevent decline in brain metabolism during aging through a combination of
estrogen-like selective estrogen receptor modulators (Genistein, Daidzein, and S-Equol) with a
mitochondrial enhancer formulation (R+Lipoic Acid and Acetyl-L-Carnitine) will sustain brain
metabolic capacity, thus delaying progression of AD. The results from this study indicate this
24
novel formulation is a potential preventative therapeutic for Alzheimer’s disease.
Key Words: PhytoSERM, mitochondria, brain bioenergetics, phytoestrogens, oxidative stress,
behavior
Introduction
We aim to develop a novel nutraceutical formulation that contains non-feminizing
estrogenic compounds and mitochondrial enhancer substrates to promote brain metabolic
activity. We predict this unique combination will counterbalance the bioenergetic system decline
in the 3xTgAD Alzheimer’s mouse model.
Alzheimer’s disease (AD) remains without an effective strategy to prevent, delay, or treat
the disease. In 2010, the World Health Organization estimated the number of persons with AD-
related dementia at 35.6 million, which is expected to triple by 2050 to over 115 million (2012).
The projected number of persons with AD in the USA by 2050 is 13.5 million, and the medical
costs will exceed $20 trillion over the next 40 years(Brookmeyer et al., 2007; Ferri et al., 2005).
The measurable socioeconomic annual costs of the disease on a global scale were estimated to
exceed $600 billion in 2010(2012). Socioeconomic data predict that significant decreases in
medical costs are possible if therapeutic development shifts to identification and prevention of
AD rather than attempts to reverse AD pathology(Lin et al., 2014). Since 1998, there have been
over 101 failed Alzheimer’s trials(Alzheimer's, 2015).
Currently available drugs offer only mild symptom alleviation(Alzheimer's, 2015). No
therapeutic strategies have demonstrated clinically significant disease-modifying benefits to halt
or reverse cognitive decline. Most of the therapeutic candidates have focused on reduction or
reversal of AD pathology based on the β-amyloid (Aβ) plaque hypothesis. Several anti-amyloid
drug candidates have failed in late-stage clinical trials (Feldman et al., 2014; Karran et al., 2011).
Despite preclinical success in cell lines and animal models, most therapeutic candidates for AD
failed to show any significant effect on cognitive function at clinical stages(Alzheimer's, 2015).
These multifactorial diseases present differently, responses to therapies also differ. For example,
25
unhealthy diet and exercise may have different impacts on individuals and require different
treatment strategies than those individuals predisposed to genetically inherited familial diseases
(Reiman et al., 2012). Sex, genetic risks, and age are important variables that should be
considered during the development stage for AD therapeutics (Swerdlow et al., 2014). The
dosing regimen, formulation, and the route of administration all have significant effects on
clinical success (Swerdlow et al., 2014). Past approaches targeting moderate and severe AD
pathology have had minimal success, in part because of the single target strategy for a
multifactorial pathology. In contrast, targeting the affected biological systems at specific stages
of disease progression may have greater likelihood of success in nonfamilial AD.
Alzheimer’s disease (AD) is a national and global epidemic. The disease population can
be divided into two subsets; familial (FAD) and late-onset (LOAD). Presenilin-1 (PSEN1)
mutations are responsible for more than 50% of familial AD (FAD) cases, which itself makes up
only 5% of the AD patient population (Yao et al., 2011). LOAD affects a much larger population
and its causes are not fully understood; they likely include a combination of environmental,
genetic, and lifestyle factors. The genetic component for LOAD had eluded researchers for many
years. One genetic risk factor discovered is the apolipoprotein E (APOE) gene on chromosome
19 (Corder et al., 1993). APOE has several alleles that are linked with differing risk factors;
APOE ε3, APOE ε2, APOE ε4(Teter et al., 2002a). APOE ε3 is the most common and holds
little influence on risk of AD. APOE ε2, the rarest form, actually seems to provide protection
against AD incidence. APOE ε4 is associated with increased risk of AD and significant earlier
age of onset. AD frequency due to APOE ε4 alleles increases from 20% (noncarrier) to 47%
(heterozygous) and up to 91% (homozygous); while the age of onset decreases from 84 to 76
then to 68 respectively(Jones et al., 2011; Koffie et al., 2012). Some of the hallmark roles of
pathogeneses that APOE4 contributes to AD include gain of toxic function and loss of
neuroprotective function; examples include increased brain atrophy, increased Aβ and Tau
aggregation, decreased glucose metabolism, decreased mitochondrial function, and decreased
lipid metabolism(Chen et al., 2007; Corder et al., 1993; Jones et al., 2011; Kim et al., 2014;
26
Koffie et al., 2012; Nathan et al., 2002; Teter et al., 2002b). The declines in the brain’s
bioenergetic system lead to downstream pathology causing cognitive impairment.
Data from other research groups indicate that mitochondrial dysfunction as well as
altered brain metabolic capacity play a causal role in the pathogenesis of Alzheimer’s (Dumont
et al., 2010b; Gibson and Shi, 2010b) (Yao and Brinton, 2012; Yao et al., 2011). Findings from
basic science research as well as epidemiological studies have suggested great therapeutic
potential of candidates targeting mitochondria for AD intervention (Andreux et al., 2013;
Brinton, 2008a; Caldwell et al., 2015; Cardoso et al., 2004b; Cavallucci et al., 2013; Dumont et
al., 2010a; Hauptmann et al., 2009a).
In multiple translational mouse models of Alzheimer’s, it has been demonstrated that
mitochondrial bioenergetic deficits precede the development of AD pathology (Yao et al., 2009)
and the decline in mitochondrial function continues to deteriorate with AD progression
(Lustbader et al., 2004). Many clinical investigations also agree, reporting antecedent decline in
cerebral glucose utilization decades prior to the onset of AD (Brinton, 2008a; Ding et al., 2013a;
Ishii et al., 1997; Nicholson et al., 2010; Swerdlow R, 1989). In AD, there is a generalized shift
away from glucose-derived energy production, which is associated with a decrease in the
expression of glycolytic enzymes coupled to a decrease in the activity of pyruvate
dehydrogenase (PDH) complex (Blass, 2000). In addition to the decline in brain glucose
metabolism, alteration in brain metabolic profile in AD is further evidenced by concomitant
activation of compensatory pathways that promote the usage of alternative substrates, such as
ketone bodies, to compensate for the decline in glucose-driven ATP generation. The divergence
of mitochondrial metabolic phenotypes between healthy aging and AD can serve as a unique
therapeutic target for multi-action: 1) to promote healthy aging and prevent AD by potentiating
glucose metabolism; 2) to delay the progression of AD by sustaining glucose metabolism with
simultaneous activation of alternative pathways, i.e. ketogenesis; 3) reduce oxidative damage to
cellular mechanisms to discard dysfunctional mitochondria; 4) improve respiration by improving
activity of specific complexes along the electron transport chain within the mitochondria (Fig. 1).
27
Figure 1: Areas to target the bioenergetic
dysfunction of the Alzheimer’s brain
preceding AD pathology and neuronal
death. These multiple target points include
1) glucose or alternative fuel for substrate
supply; 2) glycolysis, use of glucose; 3) the
citric acid cycle (TCA) cycle and electron
transport chain (ETC); 4) oxidative stress,
harmful ROS species; 5) mitophagy and
apoptosis. Mitochondrial dysfunction can
produce free radicals causing oxidative
damage; reactive oxygen species and
caspase proteases can block the
neuroprotective mitophagy pathway. (GLUT = glucose transporter; MCT = monocarboxylate transporter; PDH =
pyruvate dehydrogenase; SCOT = succinyl-coenzyme A:3-ketoacid CoA transferase) (Caldwell et al., 2015)
___________________________________________________________________________________________________________________
Almost two thirds of the Alzheimer’s population are postmenopausal women. Postmenopausal
women are at risk of cognitive decline due to diminished serum levels of ovarian sex hormones
such as estrogen following menopause (Brinton, 2008a, b). This increases the risks for cognitive
impairment and AD dementia. Though evidence suggests that estrogen therapy can potentially
counteract these changes by sustaining the brain in a proactively defensive status against
neurodegeneration, the substantial risks of oncogene expression prevent this from being a viable
therapeutic option. For this reason, translational development of selective estrogen receptor (ER)
modulators (SERMs), in particular ERβ-selective phytoSERMs as a natural approach for
potentially promoting neurological health and preventing age-associated cognitive impairment in
patients at risk of Alzheimer's disease(Yao et al., 2013).
28
SERMs are a class of drugs that act on estrogen receptors (ER). Their characteristic difference
from pure ER agonists and antagonists is that their action is different in various tissues, thereby
granting the possibility to selectively inhibit or stimulate tissue dependent estrogen-like action
(Irwin et al., 2008; Irwin et al., 2012). Our novel formulation combines the phytoSERMs with a
bioenergetic enhancer cocktail to address the multiple factors contributing to the
neurodegeneration in LOAD. A 9-month diet of our novel bioenergetic formulation aimed to
improve cognitive performance and mitochondrial respiration in a triple transgenic mouse model.
Formulation Components (Genistein, Daidzein, S-Equol, R+Lipoic Acid and Acetyl-L-
Carnitine)
In the human body there are two subtypes of estrogen receptors, alpha (ERα) and beta
(ERβ), which belong to the nuclear receptor gene family. The two receptors have a tendency to
interact with analogous DNA response elements and have similar binding affinities for an array
of ligands (Matthews and Gustafsson, 2003). The differences between the two receptors appear
in their tissue distribution and expression patterns. ERα is commonly localized and expressed in
the breast, uterus, cervix, vagina, liver, kidney and heart, whereas ERβ is localized and expressed
in the ovary, prostate, testis, spleen, lung, bladder, hypothalamus, and thymus (Kuiper et al.,
1997; Patisaul et al., 2002). Both receptors are prominently co-expressed in the central nervous
system. ERα agonists potentiate the negative effects associated with estrogens such as excessive
cell proliferation in hormone sensitive cells. In contrast to ERα agonists, it is believed that ERβ
agonists do not initiate proliferative effects (Hall et al., 2001; Matthews and Gustafsson, 2003).
PhytoSERM formulation includes equal parts Genistein, Daidzein, and S-Equol. These are two
isoflavones and one isoflavandiol are all ERβ agonists (Irwin et al., 2012; Patisaul et al., 2002;
Yao et al., 2013). ERβ agonists have been shown to inhibit the cell proliferation caused by ERα
activation (Hall et al., 2001; Matthews and Gustafsson, 2003). Previous investigations have
identified several plant-derived phytoestrogens as effective and safe alternatives to estrogen
therapy. These compounds bind at weak to moderate affinities to estrogen receptors (ERs) and
29
exert estrogenic or anti-estrogenic activities depending on the specific compound (Dixon, 2004;
Setchell, 1998; Setchell et al., 1998). A great number of both basic science research and clinical
observations have suggested that phytoestrogens could be beneficial in prevention and treatment
of multiple sex hormones-related disorders including menopausal hot flashes, breast cancer,
prostate cancer, and AD (Goetzl et al., 2007; Zhao and Brinton, 2007; Ziegler, 2004).
Compounds that target mitochondria and metabolism include small molecules derived
from natural products which function as important co-enzymes of mitochondrial bioenergetic
pathway, including R-(+)-lipoic acid and acetyl-L-carnitine benefits specific aspects of
mitochondrial function and metabolism, such as promoting glucose metabolism, enhancing
mitochondrial bioenergetics, diminishing oxidative stress, and/or inhibiting apoptosis,
respectively. We have discovered a combination of factors that enhance function of key elements
in the energetic system of the brain providing a synergistic increase in brain metabolic activity
and mitochondrial function. R-(+)-lipoic acid and acetyl-L-carnitine are two components with
well-established efficacy to promote glucose metabolism. R-(+)-lipoic acid modulates distinct
energy-redox circuits and has been reported to protect against multiple age-related
neurodegenerative diseases (Hager et al., 2007; McGahon et al., 1999; Singh and Jialal, 2008). It
is a cofactor for the mitochondrial E2 subunit of α-ketoacid dehydrogenase complexes. Lipoic
acid has been well documented to facilitate glucose uptake and metabolism (Packer and Cadenas,
2011). Further, lipoic acid facilitates the dissociation of Keap-1 from Nrf2 and thereby promotes
the transcription of endogenous antioxidant enzymes. Acetyl-L-carnitine is an endogenous
metabolic intermediate and potent antioxidant that assists transport of acetyl groups across the
inner mitochondrial membrane and is important for energy production and neurotransmitter
biosynthesis. Collectively, these are designed to potentiate glucose metabolism while
simultaneously enhancing endogenous antioxidant capacity. Lipoic acid at the proposed dosage
demonstrated significant efficacy to increase brain glucose uptake in our preliminary analyses.
The dosage of acetyl-L-carnitine is based on previous preclinical and clinical studies
(Parachikova et al., 2010; Spagnoli et al., 1991). R-(+)-lipoic acid, a component of the proposed
30
bioenergetic formulation, is a co-factor essential for multiple key bioenergetic enzymes,
including pyruvate dehydrogenase (PDH) complex, α-ketoglutarate dehydrogenase (αKGDH),
and the branched-chain oxoacid dehydrogenase (BCDH) complex. R-(+)-lipoic acid also protects
neurons against a variety of neurotoxic insults (Liu, 2008; Packer et al., 1997). These
multifactorial mechanisms of R-(+)-lipoic acid action predict therapeutic efficacy of R-(+)-lipoic
acid to promote brain bioenergetic capacity.
It has been determined that the best approach to lowering the incidence of AD is
prevention through early intervention rather than treatment of advanced cognitive damage (28,
31, 58-60). The many impacts of specific diets on cognitive health and risk of AD have been
documented in vivo and in the clinic. The 3xTgAD mouse model is a well-documented model of
AD to test an oral dietary intervention (61-64). The 3xTgAD model is also uniquely ideal for
looking at amyloid plague and tau tangle pathology development in a timely manner without
using the extremely unlikely five mutation-based model (65, 66). We hypothesize administering
3xTgAD mice with the PhytoSERM plus formulation over a 9-month period starting at a young
age will reduce AD cognitive decline, promote brain bioenergetics, and improve peripheral
metabolic system. Analysis will include brain mitochondrial function, behavior, glucose uptake
imaging, and several peripheral metabolic markers.
Significance
This study builds on our understanding of the mitochondrial bioenergetic decline in aging
and the subsequent alteration in brain metabolic profile with AD disease progression. The brain
bioenergetics are a convergent target upstream of AD pathogenic pathways which provide a
novel and unique opportunity for a combination of dietary supplementation to target
mitochondria and metabolism for maximal synergistic achievement for disease prevention and
modification. The study provides a unique scope integrating preclinical therapeutic development
with basic understanding of metabolic regulation in AD. From a clinical perspective, findings
from this study facilitate the development of potential therapeutics, particularly nutraceuticals,
31
that have great potential to prevent AD, delay the onset of the disease, and/or slow disease
progression at early stage through dietary manipulation rather than late stage medical
intervention. Financially, disease prevention or early stage modification could extraordinarily
relieve the burden on aged society. Moreover, the strategy adopted in the study to sustain and
enhance brain metabolism could be generalized to promote healthy aging and therefore benefit
the aged population to a much larger extent.
Methods
Animal treatment design
Mouse groups separated by genotype, sex, and
treatment.
Diets
Base control (AIN-93M Purified irradiated diet)
Treatment (AIN-93M modified with added
Phytoestrogens (Genistein, Daidzein, S-Equol), R-
(+)-Lipoic Acid, and Acetyl-L-Carnitine)
* Harlan Teklad Diets
Transgenic Non-Transgenic
Female Male Female Male
Treatment Groups
Cont. Phyto+ Cont. Phyto+ Cont. Phyto+ Cont. Phyto+
Total: 96
12 per group
32
In Vivo Experimental Design
To investigate the impact of PhytoSERM
Plus treatment on brain mitochondrial
function, 2-3-month-old female and male
(nonTg and 3xTgAD) mice were randomly
assigned to one of the following two
treatment groups (n=12 per group): Control
Diet AIN-93M purified diet of PhytoSERM
Plus enhanced custom AIN-93M purified
diet. PhytoSERM plus enhanced diet
formulation included 150mg/kg
PhytoSERM (equal molecular parts of
Genistein, Daidzein, and S-Equol), 6.7g/kg
Acetyl-L-Carnitine, and 1g/kg R- (+)-Lipoic
Acid. Upon completion of the 9-month
treatment, mice were sacrificed; tissues were
harvested, processed, and stored for later
analyses.
Animals
Colonies of the 3xTgAD mice strain (129S; Gift from Dr. Frank Laferla, University of
California, Irvine) bred and maintained at the University of Southern California (Los Angeles,
CA) following National Institutes of Health guidelines on use of laboratory animals. Mice were
housed on 12-hour light/dark cycles and unrestricted access to food and water. The
characterization of amyloid and tau pathologies, as well as synaptic dysfunction in this line of
mice has been described previously (Oddo et al., 2003a; Oddo et al., 2003b). To ensure the
♀
♂ 3 6 9 12
________________
Age (months)
________________
Treatment
Control
ALA/ALC
PhytoSERM
ALA/ALC + PhytoSERM
(Combination Treatment)
3xTgAD
Blood Plasma
Biochemistry
Glucose Tolerance
Triglycerides
Ketone Bodies
HOMA-IR
Energy
Metabolism
Respiratory Control Ratio
(brain and liver)
Complex I activity
Complex IV activity
P:O
Pathology Ab load
(Ab
38
, Ab
40
, Ab
42
)
Behavior Novel Object Recognition
(Discrimination Index)
33
stability of AD-like phenotype in the 3xTgAD mouse colony, we perform routine
immunohistochemical assays every 3 to 4 generations. Only offspring from breeders that exhibit
stable AD pathology were randomized into the study.
Brain tissue preparation and collection
Upon completion of the study, mice were sacrificed. Cerebellum and brain stem were removed
prior to further dissection. Cerebral cortex and hippocampus from the left hemisphere were
quickly harvested and processed for crude mitochondrial isolation. Cortical and hippocampal
tissues from the left hemisphere were harvested and stored for future protein analysis. The left
hemispheres were quickly harvested and processed immediately for crude mitochondrial
isolation to prepare for mitochondrial respiration experiments using Seahorse Biosciences eXF96
Flux Analyzer.
Peripheral Blood Studies Take suborbital blood for triglyceride, ketone, and insulin testing at
baseline and throughout the dosing with last collection at sacrifice. Mice are fasted overnight for
14 hours before blood draw. These peripheral blood markers will determine the metabolic profile
of the transgenic and non-transgenic animals including the treatment effects. This blood will be
taken from behind the eye by capillary tube into a heparin coated collection vial. The vial will
then be spun down at 3000g for 30 mins. Plasma will be removed and kept in a -80F freezer.
Cayman Chemical ELISA kit will be used to determine plasma triglyceride and ketone body
levels. Alpco ELISA kit will determine insulin plasma levels. These ELISAs preformed as
described in (Klosinski et al., 2015).
Glucose Tolerance Testing Perform glucose tolerance tests (GTT) at multiple time points
including baseline and near sacrifice. Mice are fasted over night before this procedure 14 hours.
The GTT tests were performed in 10 mouse staggered sets. Blood is taken from the tail vein.
After baseline measurement of glucose an IP injection of glucose (µl) equivalent to 4xgrams of
34
body weight. Example, a 25-gram mouse received 100ul of glucose. Following blood samples
would be taken after 5, 15, 30, 60, 90, and 120 mins after injection. Blood drops were measured
using the Freestyle Lite glucose monitoring system and freestyle light glucose strips lot code: 16
(Ding et al., 2013a; Ding et al., 2013b).
Novel Object Recognition Behavior of the mice is determined using novel object recognition
testing. This experiment was done in collaboration with the Jakowec lab instructed by graduate
student Daniel Stefanko. The object recognition task consisted of a training phase and a testing
phase (Gorton et al., 2010). Before training, all mice were handled 1–2 min a day for 5 d and
were habituated to the experimental apparatus 15 min a day for 3 consecutive days in the absence
of objects. The experimental apparatus was a white rectangular open field (30 x 23 x 21.5 cm).
During the training phase, mice were placed in the experimental apparatus with two identical
objects (150 ml beakers, 1-inch circumference x 1.5-inch height; large blue Lego blocks, 1 x 1 x
2 inches) and were allowed to explore for either 3 or 10 min. The objects were thoroughly
cleaned between trials to make sure no olfactory cues were present. Retention was tested at 90
min for short-term memory and 24 h for long-term memory. During these retention tests, mice
explored the experimental apparatus for 5 min in the presence of 1 familiar and 1 novel object.
The location of the object was counterbalanced so that one-half of the animals in each group saw
the novel object on the left side of the apparatus, and the other half saw the novel object on the
right side of the apparatus. All training and testing trials were videotaped and analyzed by
individuals blind to the treatment condition and the genotype of subjects to determine the amount
of time the mouse spent exploring the novel and familiar objects. A mouse was scored as
exploring an object when its head was oriented toward the object within a distance of 1 cm or
when the nose was touching the object. The relative exploration time was recorded and
expressed by a discrimination index [D.I. = (tnovel - tfamiliar)/(tnovel + tfamiliar) x 100%]. Mean
exploration times were calculated and the discrimination indexes between treatment groups were
compared (Gorton et al., 2010).
35
FDG-PET Imaging Brain glucose metabolic function is determined by Fluorodeoxyglucose
(18F)- Positron Emission Tomography (FDG-PET). 6 animals from each transgenic group were
tested. FDG-PET scans were taken 20 mins after injection of radioisotope and cross laid with CT
scan, so the regions of interest could be identified. Data for eyes and cerebellum were removed
for analysis.
Mice will be maintained under anesthesia during microPET and microCT scans with 2–2.5%
isoflurane in oxygen. Anesthesia is the standard procedure for small animal imaging to ensure
that body movement does not occur which would nullify the images acquired.
Scans were performed in an imaging chamber equipped with a nose cone for anesthesia delivery
and heating pad for body temperature control. MicroPET imaging was performed with a
microPET R4 rodent model scanner (Concorde Microsystems Inc, Knoxville, TN) and micro CT
imaging was performed on MicroCAT II tomography (Siemens Preclinical Solutions, Knoxville,
TN). In our mouse microPET scan, the impact of skull thickness on the signal is very small. For
microPET imaging (FDG: 511 keV), the linear attenuation coefficients of bone and water are
0.192/cm and 0.096/cm. The skull thickness of the mouse is usually 0.5 mm, and the attenuation
factor is e
-0.192x0.05
= 0.990. If we have 0.5 mm of soft tissue, the attenuation coefficient of which
is similar to water, this factor becomes e
-0.096x0.05
= 0.995. Thus, the difference in attenuation
coefficient between skull and soft tissue is smaller than 1% ((0.995–0.990)/0.990 = 0.51%).
Mice were injected intravenously via the tail vein with radiotracer [
18
F] Fluoro-2-deoxy-2-D-
glucose (FDG, 200 µCi, 100 µL). The dosage of FDG is Radioactive dose was determined prior
to injection by radioisotope dose calibrator (Capintec, CRC-712M). At 40 min post-injection of
FDG, each mouse was positioned in the MicroPET scanner in the center of the 10.8 cm
transaxial and 8 cm axial field of view (FOV). Brain microPET data were collected for 10 min
followed by a 10 min microCT scan for the purpose of co-registration.
Co-registration of microPET and microCT data was performed using the AMIDE software
package (http://amide.sourceforge.net/). After co-registration of the PET and CT images, ROI
36
(region of interest) was defined and used to measure the radioactivity concentration in brains.
Decay correction was used to adjust the actual radioactivity dosage injected (Actual radioactivity
dosage at time of injection = Initial radioactivity × e
-ln2x(T/110)
, T = T minutes between injected
time point and initial time point) (Ding et al., 2013a).
Mitochondrial Isolated Respiration Two weeks after FDG-PET animals are sacrificed brain,
blood, several organs, and tissues are collected. Half the brain and liver are stored for further
experimentation. The other halves are immediately homogenized in separate tubes in
mitochondrial isolation buffer. Mitochondrial isolation is then preformed on brain and liver and
the isolated mitochondria are plated into BioSciences SeaHorse plates to measure mitochondrial
oxygen consumption under difference conditions to obtain OCR normalized using an RCR
(relative oxygen consumption rate) score. Brain mitochondria isolation, the XF 96 Flux Analyzer
is used for this measurement. 3xTgAD mice are euthanized and the brains are rapidly dissected
on ice. Cerebellum and brain stem are excluded for mitochondrial isolation. Whole-brain
mitochondria are isolated as previously described (Yao et al., 2009). Briefly, the brain was
rapidly minced and homogenized at 4 °C in mitochondrial isolation buffer (MIB) (pH 7.4),
containing sucrose (320 mM), EDTA (1 mM), Tris-HCl (10 mM), and Calbiochem's Protease
Inhibitor Cocktail Set I (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride 500 µM,
aprotonin 150 nM, E-64 1 µM, EDTA disodium 500 µM, leupeptin hemisulfate 1 µM). Single-
brain homogenates are then centrifuged at 1500g for 5 minutes. The pellet is resuspended in
MIB, rehomogenized, and centrifuged again at 1500g for 5 minutes. The postnuclear
supernatants from both centrifugations are combined, and crude mitochondria are pelleted by
centrifugation at 21,000g for 10 minutes. The resulting mitochondrial pellet is resuspended in
15% Percoll made in MIB, layered over a preformed 23% per 40% Percoll discontinuous
gradient, and centrifuged 31,000g for 10 minutes. The purified mitochondria are collected at the
23% per 40% interface and washed with 10-mL MIB by centrifugation at 16,700g for 13
minutes. The loose pellet is collected and transferred to a microcentrifuge tube and washed in
37
MIB by centrifugation at 9000g for 8 minutes. The resulting mitochondrial pellet is resuspended
in MIB to an approximate concentration of 1 mg/mL. The resulting mitochondrial samples are
used immediately for respiratory measurements and hydrogen peroxide production. During
mitochondrial purification, aliquots are collected for confirmation of mitochondrial purity and
integrity following a previously established protocol (Irwin et al., 2008). Mitochondrial
respiration is measured using the Seahorse XF-96 metabolic analyzer. Briefly, purified
mitochondria are determined for protein concentration and resuspended into 1X mitochondrial
assay (MAS) solution (70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2
mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylene glycol
tetraacetic acid, and 0.5% [wt/vol] fatty acid free bovine serum albumin, pH 7.2 at 37 °C) 4 µg
of mitochondrial samples in 25 µL 1X MAS buffer are seeded into each assay well.
Mitochondrial samples are spun down at 2000g for 10 minutes and then supplemented with 75
µL 1X MAS buffer with substrate (glutamate/malate 5 mM). Metabolic flux cartridges are
loaded with the following reagents in 1X MAS: port A: adenosine diphosphate (ADP) (20 mM);
port B: oligomycin (30 µg/mL); port C: FCCP (carbonyl cyanide-4-(trifluoromethoxy)
phenylhydrazone) (28 µM); and port D: rotenone (16 µM) + antimycin (70 µM). Injection
volumes for all ports are 25 µL. Mitochondrial respiration is measured sequentially in a coupled
state with substrate present (basal respiration, state 4 respiration), followed by ADP-stimulated
state 3 respiration after port A injection, (phosphorylating respiration in the presence of ADP and
substrates). Injection of oligomycin in port B induces diminished respiration and the subsequent
injection of FCCP in port C induces uncoupler-stimulated maximal respiration, while the final
injection of rotenone and antimycin in port D results in nonoxidative phosphorylation–related
residual oxygen consumption OCR residual. Respiratory control ratio value is calculated as the
ratio of oxygen consumption rate (OCR) at state 3 respiration over OCR at state 4 respiration:
respiratory control ratio (RCR)= OCRstate3/OCRstate4.
Further studies using isolated mitochondrial tested for complex I and complex IV activity.
Complex I and complex IV activity are assessed in isolated mitochondria (20 µg) using Rapid
38
Microplate Assay kits (MitoSciences, Eugene, OR, USA) following the manufacturer's
instructions.
These activity tests are followed up with mitochondrial genetic copy number experiment to
determine if number or mass of mitochondria have changed. Copy number was determined by
the ratio of COXII to B-globin genes. This test controls for mitochondrial volume to determine
relative activity change.
AD Pathology Amyloid peptides will be extracted from the remaining 3xTgAD hippocampus
halves. Using a MSD Kit targeting Aβ38, Aβ40, and Aβ42. Hippocampal Aβ levels: Soluble and
insoluble Aβ concentrations will be quantified using MSD 96-well multi-spot 6E10 Aβ triplex
ultra-sensitive assay kits (Meso Scale Discovery, US). Flash frozen unilateral hippocampus will
be homogenized in Neuronal Protein Extraction Reagent (Thermo Scientific, MA) with 1X Halt
Protease Inhibitor Cocktail and EDTA in a Bullet Blender using 0.5 mm glass beads (Next
Advance, NY). Homogenates will be centrifuged at 22,000 x g at 4 °C for 20 min. The
supernatant will be transferred to a new tube, representing the soluble fraction. Next, 50 µL of
70% formic acid will be added to the remaining pellet, homogenized, and neutralized by adding
750 µL of 1 M Tris HCl. The homogenate will be centrifuged at 22,000 x g for 20 min and the
supernatant will be transferred to a new tube, representing the insoluble fractions. The protein
concentration of the soluble fraction will be measured using a BCA Protein Assay Kit (Pierce,
IL). Standard curves will be established: Aβ38, 0-3000 pg/mL; Aβ40, 0-10,000 pg/mL; Aβ42, 0-
3000 pg/mL. 50 µg of total protein will be loaded into each well; for insoluble fractions,
normalize to total soluble protein. A Sector Imager plate reader will quantify Aβ concentrations
with reference to the standard curves and expressed in pg/mL.
Statistics Statistically significant differences between groups were determined by one-way
ANOVA followed by a Newman–Keuls post-hoc analysis. The Prism software by Graphpad was
used to preform statistical tests.
39
Results
Results from studies using the phytoSERM plus formulation in 3xTgAD model. Figure 2 shows
glucose tolerance testing (GTT) and peripheral blood marker data in both male and female
3xTgAD mice. Both male and female 3xTgAD mice treated with phytoSERM plus had
significantly improved glucose metabolism compared to the control diet (Figure 2A).
0
10000
20000
30000
40000
50000
60000
Male NonTg Female Non Tg Male 3xTgAD Female 3xTgAD
AUC glucose tolerance Test
Control Diet PhytoPlus Diet
40
Figure 2: Peripheral Metabolism.
A. Area under the curve (AUC) for glucose tolerance testing. The AUC indicates level of
glucose present in the blood plasma over a specific time course of 2 hours; baseline measure
at 3months of age, control and phytoplus diet measures at 11-12 months of age.
B. Peripheral triglyceride and ketone body levels in peripheral blood plasma P-values:
*<0.05, **<0.01, ***<0.001
GTT was performed at 4 time points the AUCs of each time points show a gradual improvement
over time inn glucose tolerance (SF2). Additionally, both male and female 3xTgAD mice treated
with phytoSERM plus showed decreased levels of peripheral triglycerides and ketone bodies in
blood plasma (Figure 2B).
The 3xTgAD mouse model cognition was tested using novel object recognition a gold standard
for hippocampal function. The test compared phytoSERM plus treated to control diet along with
WT animals fed both control and phytoSERM plus diet. Both female and male 3xTgAD on
control diet preformed significantly below the WT animals. 3xTgAD mice treated with
phytoSERM plus did not should the cognitive decline and infect were statistically equivalent to
WT performance (Figure 3).
41
Figure 3: Novel Object Recognition Data.
All mice at 10-11 months of age; (n=8-10). Both female (A) and male (B)
3xTgAD mice on control diet show significantly decreased cognitive recall as
compared to the WT mice. However, 3xTgAD mice on phytoSERM plus diet
demonstrate statistically equivalent cognitive performance. P-values: *<0.05,
**<0.01, ***<0.001.
The results for the static FDG-PET imaging study on 3xTgAD on chronic phytoSERM plus diet
showed no improvement on brain glucose utilization. However, data did show a significantly
higher FDG percentage uptake for females over males (SF5).
3xTgAD mice on phytoSERM plus were tested for whole brain mitochondrial respiration. The
level of respiration was measured using RCR described in the methods section. Female 3xTgAD
mice receiving phytoSERM plus improved mitochondrial respiration rate in the brain by
approximately 77% (Figure 4C). However, the formulation did not improve male mitochondrial
respiration (Figure 4B). It is important to note the average baseline RCR for 3xTgAD males was
significantly higher than that of females (Figure 4A). The results of this brain mitochondria
respiration were confirmed by elisas measuring complex I and complex IV activity in isolated
mitochondria along with measuring mitochondria DNA copy numbers to checking for
mitochondrial biogenesis (SF3, SF4).
42
Figure 4: Mitochondrial Respiration of the Whole Brain.
Isolated mitochondria from the brain were analyzed using the Seahorse Biosciences 96XFe Flux
Analyzer to measure mitochondrial respiration. A. RCR values of baseline male and female
3xTgAD. B. Relative Male 3xTgAD RCR for control and PhytoPlus. C. Relative Female
3xTgAD RCR for control and PhytoPlus. Respiratory control ratio (RCR) value is calculated as
the ratio of oxygen consumption rate (OCR) at state 3 respiration over OCR at state 4 respiration:
respiratory control ratio (RCR)= OCRstate3/OCRstate4. P-values: *<0.05, **<0.01, ***<0.001.
Pathology for the 3xTgAD mouse model was measured using the mesoscale discovery amyloid
beta peptide quantitative elisa kit. Data showed significantly higher levels of all three amyloid
beta peptides for females compared to male 3xTgAD mice (Figure 5 and SF1). Males on
phytoSERM plus showed a decrease in 38 and 40 A-beta load compared to control diet. No
statistically significant dietary impact was seen on females nor males for A-beta 42.
43
Figure 5: Amyloid Beta Peptide Load in the Hippocampus.
Data from Mesoscale Discovery Kit to detect amyloid beta peptides 38, 40 and 42. All mice were 3xTgAD and
between 11-12 months of age. Data shows males on phytoSERM plus have significantly lower AB38 and
AB40. There was no treatment effect for AB42 shown in supplemental figures. P-values: *<0.05, **<0.01,
***<0.001
Discussion
Alzheimer’s (AD) remains without an effective strategy to prevent, delay, or treat the disease.
In 2010, World Health Organization estimated the number of persons with AD related dementia at
35.6 million, which is expected to triple by 2050 to over 115 million (2012). The projected number
of persons with AD in the US by 2050 is 13.5 million, and the medical costs will exceed $20 trillion
44
over the next 40 years (Brookmeyer et al., 2007; Ferri et al., 2005). The measurable socioeconomic
annual costs of the disease on a global scale were estimated to exceed $600 billion in 2010 (2012;
Feldman et al., 2014). Socioeconomic data predict that significant decreases in medical costs are
possible if therapeutic development shifts to identification and prevention of AD rather than attempts
to reverse AD pathology (Lin et al., 2014).
Study Diagram: The figure outlines which experiments were done to determine the effectiveness
of treatment on the different groups of 3xTgAD mice using markers for blood biochemistry,
energy metabolism in the brain, pathology, and behavior.
In the current study, we demonstrated that the PhytoSERM Plus formulation potentiated
mitochondrial respiration in the brain, improved peripheral metabolic markers, and protected
against cognitive decline. Experiments in this study confirm previous results linking components
to proposed mechanism of action. The unique combination of these components positively
impacts glucose metabolism, mitochondrial function, brain pathology, and cognition in the
3xTgAD model. The formulation had several sex specific treatment responses. This provides
evidence that sex differences need to be addressed at the preclinical drug development stage.
45
PhytoSERM Plus formulation as an impact on peripheral blood plasma levels of triglycerides,
ketones, and tolerance of glucose. Lower levels of triglycerides and ketones could be due to an
increase in insulin production, which would also result in the increased rate for metabolism of
glucose. Rather than an increase it could also be improving the binding affinity with insulin
receptors. These peripheral impacts have an overall positive health impact on the brain.
PhytoSERM Plus had a preventative or neuroprotective effect on 3xTgAD mice cognitive recall
through novel object recognition. Treated 3xTgAD animals earned discrimination index scores
statistically equivalent to wild type mice. While untreated 3xTgAD mice preformed significantly
lower than the untreated/treated wildtype and treated 3xTgAD mice. This suggests that the
PhytoSERM Plus formulation is neuroprotective as the 3xTgAD mice on chronic treatment did
not experience cognitive decline.
PhytoSERM Plus had a positive impact on female 3xTgAD whole brain mitochondrial
respiration. It did not statistically improve nor hinder male 3xTgAD mice mitochondrial
respiration. Male 3xTgAD had significantly higher mitochondrial RCR scores than females at
baseline. This suggests that a possible explanation for the lack of treatment response in males is
because they were already operating at maximum respiratory capacity. Another possibility is that
the phytoestrogens in the formulation have a larger impact on the female mice as they move
through perimenopause, a state previously linked with mitochondrial dysfunction.
PhytoSERM Plus had no treatment effect on brain glucose metabolism as seen through static
FDG-PET imaging. However, females had significantly higher percentage FDG uptake than
males as seen in supplemental figures. It is possible the glucose metabolism impacts in the
periphery of the formulation combined with the improvement of mitochondria within the brain
result in lower need for glucose uptake for a more efficient bioenergetics system. Further studies
are needed to determine glucose uptake in the brain using a dynamic PET imaging approach.
PhytoSERM Plus had significant impact on reducing incidence of amyloid beta peptides 38 and
40 in male 3xTgAD hippocampal tissue. Diet had no impact on female pathology and no impact
on pathology for either gender for peptide 42. Female 3xTgAD amyloid beta 38, 40, and 42 were
46
all significantly higher than males at the same age of development, 11-12 months. This suggests
when studying pathology development in the 3xTgAD mouse model sex must be separated and
even possibly studies at different time points. The female mice showed much higher variability
in amyloid load than the males. Future studies should also investigate possible reasons for this
the high variability of pathology in female 3xTgAD mice over males and sectioning to see if this
variability trend extends beyond the hippocampus.
Females responded differently than males to the PhytoSERM Plus in several studies, which was
expected due to the nature of phytoestrogenic action on perimenopausal stage. Due to the
diversity in animals’ phenotype certain experiments yielded high variability. This variability
could suggest a responder and non-responder theory. This would propose certain animals’
bioenergetic or metabolic systems responded to the treatment, while some did not due to other
factors. These Factors could include peripheral organ health, environmental enrichment, genetic
abnormalities, or social hierarchy changes in cages during treatment. This is often the case in the
clinic; certain patients might respond to a particular drug and others do not without the cause of
this diversity readily apparent. More recent evidence has suggested some of these treatment
response differences can be due to exercise, diet, mood, and more.
This study shows promise for a novel nutraceutical formulation to help prevent AD. However, it
has also introduced new questions that must be answered during this formulation future
developments. Future studies developing the nutraceutical brain bioenergetic formulation might
further benefit from the inclusion of unsaturated fatty acids such as DHA and EPA due to the
high volume of acetyl-L-carnitine which is proposed to be acting as a major transporter of fatty
acids to the TCA cycle to produce more ATP to counter balance lower ATP production from a
dysfunctional mitochondrial electron transport chain. Though no major health concerns were
seen in this study with chronic dosing in mice; future studies should investigate the possible
metabolites being processed by the liver and kidneys. These studies could take animals’
excretions and test for metabolites with mass spectrometry or HPLC. Further experimentation on
peripheral organs involved with compound clearance including liver and kidney for safety
47
testing. The behavior aspects of this study can be expanded using the Morris water maze, fear
conditioning test, and food-based T-maze. Further tests with heart tissue could be important to
spotting effects on coronary and vascular health. Further genetic studies can investigate genes
encoding enzymes (Hmgcs1, Hmgcr, Fdft1, and Soat1) and transcription factors (Srebf1, CNBP
and Hbp1) involved in cholesterol biosynthesis along with phytoSERM impact on genes
involved in neuroprotective and immunosuppressive pathways mediated by estrogen (Esr1,
Grb2, Ncoa3, Phb2, Ctbp2, Gtf2e1, Polr2f, and Rbm9) (73). Many diet studies have also looked
at effects of exercise for AD; an additional exercise treadmill study could determine if exercise
can be an additive benefit used in clinical drug studies. Targets related to diabetic metabolism
should be investigated mainly focused on insulin resistance due to previous work with lipoic acid
in the 3xTgAD model (67).
One of the widely accepted therapeutic strategies to prevent Alzheimer’s is diet and exercise.
The systems-wide neuroprotective benefits of caloric restriction and exercise include activation of
adaptive cellular stress responses, enhancement of DNA repair, promotion of mitochondrial
biogenesis, and induction of neurotropic factors (Mattson, 2012). Previous preclinical studies using
multiple forms of caloric restriction led to reduced abdominal fat mass, decreased cellular oxidative
damage and pro-inflammatory cytokines (Harvie et al., 2011; Johnson et al., 2007; Redman and
Ravussin, 2011; Weiss and Fontana, 2011), and improved learning and memory function (Krikorian
et al., 2012).
Exercise has also been investigated for its direct benefits for AD patients. Exercise activates a
full systems effect including promotion of hippocampal neurogenesis, reduction of brain
inflammation, and increased PGC1a levels, mtDNA, proteins in ETC, and neurotropic factors
(Cotman and Berchtold, 2002; E et al., 2013a; E et al., 2013b; Navarro et al., 2004; Steiner et al.,
2011; van Praag et al., 2005). Interestingly, a recent study demonstrated that the benefits of exercise
were associated with elevated lactate levels and could be partially replicated by treatment with lactate
(E et al., 2013b).
48
Another lifestyle strategy for AD prevention is the Mediterranean diet (MeDi). Medi,
opposed to the Western diet, is characterized by the abundant consumption of plant foods such as
vegetables, fruits, breads, potatoes, legumes, nuts and seeds; olive oil as the source of fat; moderate
amounts of dairy, fish, poultry, and eggs; low intake of red meats; and wine during normal meals
(Bach-Faig et al., 2011; Keys et al., 1986; Willett et al., 1995). The nutrients within the MeDi
influence biological mechanisms affecting vascular, antioxidant, and inflammatory pathways (Alles
et al., 2012; Serra-Majem et al., 2006). MeDi was demonstrated to reduce risk of heart disease,
decrease markers of oxidative stress, and lower inflammatory markers; hence MeDi adherence might
delay age related cognitive decline (Dai et al., 2008; Gaskins et al., 2010; Giugliano and Esposito,
2008; Sofi et al., 2010). MeDi has shown a trend of benefiting cognitive function when assessed in
population studies in seven different countries (Feart et al., 2013). Mechanistically, the cognitive
benefits of MeDi have been attributed to the synergistic interactions between antioxidants, B
vitamins, omega-3 fatty acids, and other compounds (Feart et al., 2012; Jacobs et al., 2009).
Questions remain about whether the benefits of the MeDi could be attributed to specific ingredients
rather the complete diet (Feart et al., 2013). Investigations on the mechanisms of MeDi action could
identify key active ingredients that can be further developed into therapeutics for AD prevention and
treatment.
Other than these lifestyle strategies there are also systems approaches for improving
bioenergetics and mitochondrial function. One of the most investigated therapeutics in women is
estrogen-containing hormone therapy. 17-β-estradiol (E 2) activates multiple signaling pathways in
the brain including mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K),
G protein regulated signaling, c-fos, protein kinase C (PKC), and Ca
2+
influx, which all are
connected to neuronal function and survival (Brinton, 2008a; Rettberg et al., 2014). The ovarian
hormone loss in menopause has been linked with cognitive decline that increases the risk for AD
(Brinton, 2008a, b; Dixon, 2004; Hammond, 1994; Rettberg et al., 2014; Setchell, 1998; Yao et al.,
2013; Zhao and Brinton, 2007). E 2 treatment induced a significant increase in expression of glucose
transporters (Shi and Simpkins, 1997) and promotes aerobic glycolysis by increasing gylcolytic
49
enzyme activity of hexokinase, phosphofructokinase and pyruvate kinase (Kostanyan and Nazaryan,
1992). Further, E 2 activates PDH, enhances activities of the ETC complexes and promotes ATP
generation. The neurological benefits of E 2 are further enhanced by suppression of the oxidative
stress via enhanced antioxidant capacity. E 2 also reduces AD pathology by both decreasing the
production and increasing the clearance of Aβ species (Rettberg et al., 2014). The systems-biology of
E 2 action in the brain has led to the design and development of brain specific candidates of selective
estrogen receptor modulators (SERM) that activate the systems level of estrogenic mechanisms in the
brain without the proliferative side effects in the periphery (Dixon, 2004; Setchell, 1998; Yao et al.,
2013; Zhao and Brinton, 2007).
Clioquinol is a therapeutic candidate for treatment of AD with bioenergetic system benefits.
Acting as a chelator for copper and zinc ions, clioquinol had significant success in preclinical studies
with transgenic AD mice. Binding of metal ions is required for Aβ aggregation and Aβ-induced free
radical release in mitochondria (Adlard et al., 2008). A second-generation clioquinol molecule,
PBT2, is in phase II clinical trials (ClinicalTrials.gov identifier: NCT00471211). PBT2 reportedly
decreased Aβ levels and improved performance on two cognitive function tests (Lannfelt et al., 2008;
Ritchie et al., 2003). In addition to metal chelation, PBT2 has a second mechanistic action to increase
Aβ clearance, increasing activity of matrix metalloproteases including neprilysin, insulin degrading
enzyme, and tissue plasminogen activator (Lannfelt et al., 2008; Ritchie et al., 2003).
Souvenaid was originally developed to improve nutrient deficiencies common in AD patients
and contains high doses of Omega-3 fatty acids Eicospentaenoic acid (EPA) and Docosahexaenoic
acid (DHA) (Mi et al., 2013). The formulation also acts as a ketogenic dietary supplement with high
fat content to provide ketone bodies to the brain. The formulation also contains antioxidants,
vitamins A, C, E, riboflavin, and folic acid, selenium and ions required for membrane potential
balancing and mitochondrial function including sodium, potassium, chloride, calcium, and zinc
(Scheltens et al., 2012). In a 24 week double blind gender balanced clinical trial with 259 mild AD
patients, Souvenaid significantly improved memory and synaptic connectivity measured by
electroencephalography (Scheltens et al., 2012). The age range for patients was 51-89 years with a
50
mean of 74 years. However, in a clinical trial in moderate to severe AD patients, no significant
improvement with associated with Souvenaid treatment (Mi et al., 2013; Scheltens et al., 2012),
suggesting that the intervention window is early in AD progression.
Summary and Concluding Comments
The bioenergetic system is a complex network of pathways responsible for energy production
required for neurological function and health. Current preventive strategies to target brain
mitochondria focus on antioxidants, anti-apoptosis agents, and bioenergetic enhancement. Several of
these strategies have shown efficacy in preclinical investigations, however most interventions have
not translated to success in preventing, delaying, or reversing cognitive decline in clinical
investigations.
A large body of evidence indicates that targeting one component of a neurobiological system
does not create a course of correction nor does it reverse a system failure. For example, targeting
oxidative stress does not alleviate the glucose hypometabolism or mitochondrial dysfunction, which
are likely to be the primary failure points of the system from which oxidative stress emerges.
Attempts to target the bioenergetic system in AD faced the challenge of a dynamic adapting system
that requires biomarkers specific to the bioenergetic state and precision therapeutics that target the
bioenergetic phenotype in the window of opportunity.
The prodromal state is a promising window for new strategies for AD prevention (Figure1).
This window of opportunity is likely to be addressed through a combination of dietary supplements
and nutraceuticals. Dietary supplements are defined as products that intend to supplement diet
containing one or more of several dietary ingredients: vitamins, minerals, herbs, amino acids,
concentrates, metabolites, or combinations of such (Kalra, 2003). Nutraceuticals, which may include
one or many of the components in dietary supplements, intend to aid in prevention or treatment of
disease or disorder (Kalra, 2003). Nutraceuticals hold promise as effective modifiers of multifaceted
cellular pathways that are defective in the prodromal state of AD. Numerous vitamins and natural
compounds elicit effects on specific targets of the bioenergetic system in the brain; some
51
micronutrients may offset the deficiencies often associated with early AD (Lopes da Silva et al.,
2014; Mi et al., 2013). Natural compounds combined into a synergistic formulation could provide an
effective nutraceutical-based mode of prevention for AD and other neurodegenerative disorders.
Effective strategies that target the prodromal AD window could combine the benefits of bioenergetic
system enhancers to promote glucose metabolism, reduce oxidative stress and sustain normal
mitophagy. The systems-based therapeutic strategy to prevent early bioenergetic deficits in the brain
could have a major impact on future incidence of Alzheimer’s disease.
52
Supplemental Figures
SF1:
53
SF2:
*
*
54
SF3:
SF4:
55
SF5:
56
CHAPTER 3
APPLICATIONS FOR IMPACTING PARKINSON’S THROUGH EXERCISE, CLINICAL
LOG
Background
Parkinson's disease (PD) is a neurodegenerative disorder that leads to loss of
dopaminergic neurons in the substantia nigra pars compacta of the brain. Symptoms progress
slowly over the years with disease. The diversity of the disease leads to a wide range of symptom
profiles between patients. These PD symptoms include but are not limited to resting tremor,
bradykinesia, limb rigidity, gait and balance problems. The etiology of PD remains largely
unknown and still under investigation. There are no cures. However, several medications,
primarily different forms of dopamine replacement, are significantly helpful in managing the
motor (tremor, bradykinesia) symptoms. Cognitive problems may also occur in PD, although
there are less effective treatments available for thinking issues. Surgical options, primarily
consisting of Deep Brain Stimulation (DBS) is considered a treatment option for those
individuals suffering from DA related side effects, such as dyskinesia. While the etiology of
neuronal loss in the substantia nigra is poorly understood, one potential cause may be due to
Lewy bodies accumulation in substantia nigra neurons. Besides research focused on finding a
cure to prevent further cell loss, other studies are exploring biomarker opportunities for PD that
can lead to earlier diagnosis and more personalized therapeutic strategies. No current
medications halt or slow disease progression, however the most useful therapeutic intervention
maybe accessible to all patients to certain degree. Exercise! Exercise in animal models has been
shown to improve symptoms and repair cognitive and motor circuits impaired by DA loss.
57
In office rotation
I completed my clinical rotation in Neurology with Dr. Giselle Petzinger. Dr. Giselle
Petzinger is a Neurologist at the Keck Medical Center specializing in exercise research and its
impact at several levels in therapeutic strategies for neurodegenerative diseases. Parkinson’s
patients make up the majority of who she works with in the office. This rotation was of interest
and very applicable to my current research using Huntington’s mouse model. My current work is
investigating the metabolic pathways impacted by chronic exercise in a late stage disease state.
Exercise as an intervention has already shown very impressive outcomes both physically and
cognitively in both animal models and in the patients through the clinic.
In office daily process
The following is the process that I observed regarding Dr. Petzinger’s clinical
assessments with her patients. Besides reviewing their symptoms and reported changes from
their previous visits, she reviews their medicines, including timing of their medications and any
adverse effects from medications. She performs a neurological examination to evaluate their
Parkinsonian features and reviews this with the patient. She often reviewed the notes of the
patient’s previous visit before seeing the patient in the room. Often it was important to look at
blood pressure numbers and any changes to meds since the previous visit. When the patient
enters the room, she would greet them and open with some comfortable conversation topics
usually revolving around an interesting job they might have had before retiring. Next is a
discussion on the meds. Usually patients were on some form and dose of a dopamine
replacement compound. Medication may also include a drug to help cognitive symptoms such as
Namenda. Patients who had autonomic problems, such as low blood pressure may also be on a
medicine to increase pressure, such as fluodrocortisone. Many PD patients have autonomic
issues with a significant drop in blood pressure when going from sitting to standing due to
insufficient constriction of blood vessels and lack of signaling from the parasympathetic system.
58
This can be very dangerous and lead to falls leading to other major injuries (hip fractures, etc.)
Most patients are in their “on” state while in the office, meaning they are on dopamine
replacement and are responding. Rigidity is checked by assessing tone in the limbs.
Bradykinesia is assessed by performing repetitive movements such as Finger taps and hand
opening and closing, to assess amplitude. She looks for amplitude rather than just speed.
Bradykinesia is both low amplitude and speed. Most PD patients can do these hand movements
quickly but on repetition of movement the amplitude diminishes. After the checking amplitude
and tone, Dr. Petzinger will check their limb strength. It important to note that their limb strength
is typically normal in PD, since PD is a problem with the Basal Ganglia and not the Motor tracks
(Upper Motor Neuron). Finally, she watches them walk. The patient would stand and then walk
forward and sometimes even backward in the hallway. She would observe for slow pace along
with small amplitude, either short stride (distance between steps) or decreased arm swing.
Anxiety may also be a significant contributor to fear of falling. Dr. Petzinger would often remind
patients to swing the arms and take larger steps to promote larger amplitude movements and
make gait or walking more normal. Lastly, at the end of the visit she would discuss any other
concerns that the patient or caregiver, if present, had. Sometimes this would be a spouse who
would ask about active dreaming or restless sleeping habits. Other times it may be more severe
with a caregiver needing to work and not having someone else to help care and watch more
heavily demented patients during the day. Another issue often discussed at the end of the visit
would be the cost of the meds. Sometimes a provider would change or policy adjustment and
certain drugs or forms were very expensive. This could usually be solved by changing the dose
or formulation. Sometimes Dr. Petzinger would recommend obtaining certain drugs from
Canada. At the end of the visit, the patients would request any refills needed and schedule a
follow up for 3-4 months, depending on any ongoing issues that needed to be followed.
59
Patient observed patterns
During my time in the clinical rotation I observed several patterns and interesting aspects
within the Parkinson’s patient population. One interesting observation was the wide range of
diverse symptoms differing between patient. Certain patients had more severe tremors and issues
walking, but they seemed to have little cognitive disfunction. On the other hand, some patients
had few problems with mobility, but moderate to advanced dementia. Patients with heavy
dementia often would have a hard time hold any conversation. Sometimes they would forget
where they were or even who was sitting right next to them. Several factors may impact thinking
in PD, and these include previous employment, education and lifestyle. Did their job require
skill, were they highly educated, and did they have exercise routine? This leads me to my second
major observation, difference in willingness to exercise to improve their condition. Patients with
a history as a collegiate athlete or recreational weekend warrior would be willing to try any
recommended non-pharmaceutical options. Dr. Petzinger would recommend not only walking or
exercise but also changing the environment that they do these activities. So rather than running at
the gym on a treadmill, maybe run though a different park each week. The constant change in
environment keeps the brain more engaged. However, some patients would be out of shape and
require extra motivation to walk a bit each day. These patients often had more mobility issues
and cognitive issues. The willingness to participate in exercise might be linked to their emotional
state as well. Several patients had high levels of anxiety which prevented them from movement
and kept them from sleeping properly. We referred some of these patients with high clinical
anxiety to PD group therapy class here at Keck.
Key therapy and future options
The most important influencer of Parkinson’s patients progress was exercise. Based on
the patients who I was able to observe in clinic, I noted that those patients who were very
60
physically active and were engaged in regular exercise and other skill-based training activities
seemed to be less burden by the disease. Patients who exercised regularly often exhibited less
symptoms than non-active patients, even 10 years into their disease. Another important
observation was that after 3-5 years of disease it becomes very important for patients to follow
their medication schedule regularly in order to maintain their best drug response, called “best on
state”, throughout the day. Exercise seemed to help them get into the “on state” a bit better as
well. These observations support my interest in studying exercise intervention and mechanisms
that lead to these improvements in PD. For example, long-term exercise may alter the brains
metabolic and/or mitochondrial profile and promote repair. Possible blood-based bio-markers
might be mitochondrial function complex activity or biogenesis. Others may include glycolytic
markers or levels of nitrates and nitrites in the blood directly after some form of cardiovascular
activity. The primary take home regarding intervention in PD is that optimizing medication as
well as maintaining regular exercise is important.
Patient Log
1) Male 78yrs old- right handed
Parkinson’s Disease: Primary complaint- Poor Mobility
Symptom Profile: Significant mobility issue in wheel chair, motor dysfunction, diminished
amplitude with the hand and foot tap tests (more prominent on the left side of body), acting out
dreams, occasional hallucinations of specific people, low energy falling asleep often, gets
migraines with white flashes in vision, problems with incontinence at night.
Treatment Plan: Optimize dopamine replacement through the following-Added generic Parcopa
melts as fast acting rescue, lowered Ropinerole dose to help diminish the hallucinations, Dose
timing of Carbidopa / Levodopa was reevaluated starting a 6am every 4 hours with extended
61
release at bedtime and 1-2 extra tablets before going out at night especially with heavy meals.
Exercise recommended with guidance of Physical Therapist since patient is limited on mobility.
2) Male 69 yrs old- right handed
Parkinson’s Disease: Primary complaint -Cognitive Impairment and Falls
Symptom Profile: Motor functions limited but can walk, but cognitively impaired. Patient
complains of lack of concentration and forgetfulness. Balance also a large issue, multiple falls
and increased anxiety of falling. His gait is very slow and festinating, improves (faster speed and
bigger steps) after walking a bit, he looks like he is in off state. Currently on two tablets of
Carbi/Levodopa three times a day and 1 pill Artane three times. He reports he goes to gym 1.5hrs
every day and works on stationary bike.
Treatment Plan: Patient is underdosed which may explain both cognitive slowing and slow
walking. Plan to increase overall dopamine replacement by increasing his Carbi/Levodopa. This
will be done slowly, by 1 pill a day to avoid adverse effects. Patient was reluctant and clearly
doesn’t want to be taking to many meds but needs to optimize his “on” state. Also, the slightly
higher dose might help concentration. Recommendation to change workout environments and
mix up his activities to help his cognitive function. Suggestion made to increase his outside
activity and less sitting in gym. Also, recommendation given to increase exercise intensity,
biking needs to be more strenuous. Patient wasn’t getting heart rate to 90 bpm.
3) Male 64 yrs old. Right handed
Spinal Cerebellar Ataxia: Primary Complaint- Muscle spasms and dysphagia
Symptom Profile: Very active and engaged patient. Does weights and bike 3x week 20min in the
gym. Can walk but drags right leg, slight issues with speaking but has now been to speech
therapy 6 times and has list of words to practice. Growing concern over his issue with
swallowing liquids. Loss of dexterity in right hand and shoulder becoming more prominent.
62
Treatment plan: Increase dose for Carbi/Levo also consider going on gabapentin or baclofen for
muscle spasms. Change workout location to increase environmental complexity. Continue with
speech therapy for management of speech and swallowing.
4) Male 54 yrs old Right Handed
Parkinson’s disease: Primary Complaint – Stable, no complaints
Symptom Profile: Motor functions normal. Active, no resting tremor, very minimal issues with
slowness. Slight Cramping on right side in foot mostly in the morning (likely during off state).
Previous complaints of cognitive issues with creativity and focus at last visit. After 8 months on
higher Carbi/Levo dose he seems to feel more clarity and creativity coming back to his daily life.
Treatment Plan: Stay active with exercise, no changes in meds. Self-monitor cognitive changes
and times of day and dose.
5) Female 27 yrs old. Left- handed
Generalized Dystonia: Primary Complaint- Muscle pain/Muscle Contraction of Neck Muscles
Symptom profile: Severe mobility issues, wheel chair, major issue of involuntary muscled
contraction in neck muscles, primarily affecting right side. Non-English speaking (Mandarin),
used translator to communicate with the patient’s family/caregiver. Also concerning subluxation
of left shoulder. May be due to disease or previous injury.
Treatment Plan: Needs Brain MRI as part of complete workup and rule out Basal Ganglia injury.
She was given Botox injections along the neck to help with involuntary muscle contractions
related to dystonia and alleviate pain. Referral to ortho for evaluation of right shoulder. Consider
possibility of for deep brain stimulation for management of Dystonia and improved quality of
life. Dr. Petzinger started initial notation to propose patient for DBS.
6) Male 59 yrs old
Parkinson’s Disease: Primary Complaint – Urinary Urgency (frequent visits to bathroom)
63
Symptom Profile: Slight tremors at rest. Right side slightly slow than right noticed during gait
with decreased arm swing. Just had back surgery but doing well. No change in medications.
Received refills on his L-dopa meds plus PT therapy for the gait and balance work. Mood is
good, and thinking is good. Father worked on NASA space shuttles. Urinary problem likely
related to problems in autonomic nervous system, also affected in PD. Smooth muscle
contraction of urinary sphincter not strong enough, leads too frequent visits to bathroom.
Treatment Plan: Recommend more active exercise routine. Also recommended finding another
skill-based activity. Provided referral to urologist about the urinary urgency. Also readjust blood
pressure meds because noticed a significant drop in BP upon standing. Later also related to
problems with Autonomic NS.
64
CHAPTER 4
EXERCISE RESCUES MITOCHONDRIAL FUNCTION AND MOTOR BEHAVIOR
IN A CAG140 KI ADVANCED HUNTINGTON’S DISEASE MOUSE MODEL
Abstract
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an
excessive polyglutamine (CAG) expansion in the Huntingtin (Htt) gene resulting in a mutated
form of the huntingtin (htt) protein. This study was aimed at assessing the effects of long-term
exercise training (treadmill running) on mouse model of advanced Huntington’s disease
(CAG140HD), which recapitulates the progression of motor symptoms emerging at 12 months of
age. Long-term exercise training elicited a substantial increase of brain [nitrite + nitrate] levels
(surmised as nitric oxide), which resulted in inhibition of transglutaminase activity. Downstream
effects of the inhibitory effect on transglutaminase were expressed as a release of its inhibitory
effect on aconitase and subsequent increase in tricarboxylic acid-generated reducing equivalents
and mitochondrial oxidative phosphorylation complexes activity. Mitochondrial function was
further strengthened by increases in glycolysis, pyruvate dehydrogenase activity, and anaplerosis
component represented by pyruvate carboxylase. Metabolic homeostasis –regulated by the
stress-responsive Jun-N-terminal kinase (JNK) and insulin signaling was restored by the exercise
training intervention. These changes resulted in an improved physical and cognitive performance
as evaluated by the rotarod test.
Keywords: Huntington’s disease, long-term exercise training, nitric oxide, transglutaminase,
mitochondrial function, insulin signaling, skill-based learning, rotarod, aconitase, insulin
signaling
Abbreviations: HD, Huntington’s disease; PFK1/2, phosphofructokinase 1/2; PDH, pyruvate
dehydrogenase complex; TG, transglutaminase.
65
1. Introduction
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease that impacts
1 in 10,000 people. Patients phenotypically are characterized by progressive decline in cognitive
and motor functions with neuropsychiatric disturbances leading ultimately to premature death 15
to 20 years after onset of motor symptoms (Walker, 2007). The disease is defined genetically by
an excessive polyglutamine (CAG) expansion in the Huntingtin (Htt) gene on exon 1 of
chromosome 4 resulting in a mutated form of the huntingtin (htt) protein (McColgan & Tabrizi,
2018). Patients with more than 39 CAG repeats are certain to develop the disease, while lesser
forms are seen in repeat number between 36 and 39. This mutation causes neuronal dysfunction
and eventually death through several different mechanisms. The direct effects from the exon 1
mHTT fragment is most prominent, however the aggregates have significant indirect effects on
cellular proteostasis, metabolic, mitochondrial and synaptic function (McColgan and Tabrizi,
2018 ).
There are currently no cures for the disease and only mildly effective symptomatic drugs.
Though therapeutic strategies for perturbing or reversing the progression of the disease are still
under development, mounting evidence supports cardiovascular exercise is associated with
reduced risk of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s
(Chen et al., 2005; Stefanko et al., 2017). Exercise maybe impacting these diseases in a number
of ways, however it is know that continued cardiovascular exercise elevates levels of nitric oxide
in the body (Kingwell, 2000).
The HD CAG140 KI mouse model is characterized by 140 CAG repeats in exon 1 of the
mouse huntingtin gene ortholog (hdh) and has been reported to demonstrate a protracted
prodromal phase with motor features presenting after 12 months of age. The CAG140 KI mouse
is an optimal model for investigating the mechanisms of exercise-induced nitric oxide production
on disease modification and evaluating the functional outcomes linked to biochemical markers of
brain bioenergetics. Few studies have investigated the effects of exercise on HD models and
even fewer have addressed the impact on aged HD animals (Cepeda et al., 2010; Herbst and
66
Holloway, 2015 ). Previously published studies in Parkinson's disease (PD) have successfully
utilized the motorized treadmill as an intervention and mode for controlled and measurable
chronic exercise (Ji et al., 2015; Stefanko et al., 2017) as well as the benefits of this exercise
form on mood, cognition, motor function, dopamine (DA) neurotransmission and synaptic
plasticity (Kintz et al., 2017; Petzinger et al., 2007; Stefanko et al., 2017). Mitochondrial
dysfunction has been observed in HD in several forms (Altuntas et al., 2014; Chaturvedi and
Beal, 2013; Ferreira et al., 2018; Ismailoglu et al., 2014). Hence, it is important to assess the
impact of chronic exercise on the bioenergetic profile of the advanced Huntington’s disease
mouse model.
This study was aimed at characterizing the mechanisms by which chronic exercise impacted
the metabolic and bioenergetic profile of aged Huntington’s mice; these mechanisms are
centered on the enhanced levels of brain [nitrite + nitrate] (surmised as nitric oxide) caused by
treadmill training that result in changes in the metabolic profile elicited by initial modifications
of transglutaminase activity. These metabolic changes are expressed as a functional outcome that
entails skill-based learning in the rotarod test.
2. Materials and methods
2.1. Mice
For these studies we used knock-in mice that contained a chimeric mouse/human exon 1 with
140 CAG repeats inserted into the mouse gene by homologous targeting (Menalled et al., 2003).
CAG140 KI mice were produced in-house using lines descended from heterozygous pairing. The
founder mice for the colony were a gift of Drs. Michael Levine and Carlos Cepeda (UCLA) with
permission from Dr. Scott Zeitlin (University of Virginia) through a Material Transfer
Agreement. These mice were backcrossed onto the C57BL/6J background annually to maintain
vigor. Mouse genotypes from tail biopsies were determined using real time PCR (Transnetyx,
Inc., Cordova, TN). Mice were randomly assigned to one of 2 groups (i) CAG KI, n = 8 (3 males
and 5 females) and (ii) CAG KI + exercise, n = 8 (3 males and 5 females). No differences
67
between males and females HD mice have been reported; hence groups were collapsed for
analysis. 13-15 months old were used to represent moderate to advanced Huntington’s. Mice
were group housed with a reverse light cycle (lights off from 7 a.m. to 7 p.m.) and were allowed
access to food and water ad libitum. Experimental procedures were approved by the University
of Southern California’s Institutional Animal Care and Use Committee (IACUC) and conducted
in accordance with the National Research Council Guide for the Care and Use of Laboratory
Animals (DHEW Publication 80-23, 2011, Office of Laboratory Animal Welfare, DRR/NIH,
Bethesda, MD). All efforts were made to minimize animal suffering and to reduce the number of
animals used to achieve statistical significance.
2.2. Exercise Regimen
Treadmill running was initiated in HD mice at 13-15 months of age, performed on a Model
EXER-6M Treadmill (Columbus Instruments, Columbus, Ohio). The treadmill exercise protocol
was conducted based on previous publications with modifications (Fisher et al., 2004). Briefly,
in week one, mice in the exercise groups ran at a speed of 8.0 ± 0.5 m/min for 40 min and they
were closely monitored for any adverse reaction to the treadmill, inability to run, or failure to
learn the task. Exercise was started at a velocity of 10.0 ± 1.5 m/min and ran 5 times per week
for a 3-month period. Treadmill speed was gradually increased to 20 ± 1.5 m/min by the final
month. A non-noxious stimulus (metal beaded curtain) was used as a tactile incentive to prevent
animals from drifting back on the treadmill. All mice were weighed at the end of each week and
closely assessed for adverse reactions including stress; there were no abnormalities in weight.
Treadmill running was not stressful based on the evaluation of anxiety, depression, and
corticosterone levels (Gorton et al., 2010). All mice tolerated the exercise regimen, with no
dropouts.
68
2.3. Rotarod test
Behavioral testing consisted of motor performance using the accelerated rotarod. Outcome
was measured by latency to fall in seconds. Prior to the start of the Rotarod task, mice were
handled twice daily for 3 days. Mice were given ten minutes to acclimate to the behavior room
environment on the day of the task. The Rotarod machine was set to its base speed as mice were
assigned to their respective lanes. At t = 0s, the machine began its acceleration phase reaching
peak velocity at t = 24 s before decelerating. At t = 48s, the machine switched directions and
repeated the cycle. Endpoint criteria for the Rotarod task were: 1) Mouse falling off the cylinder
onto the platform 2) Mouse completing three revolutions via clutching onto the cylinder pad 3)
Mouse showed any signs of pain or distress. For criteria 1 and 2, the time was recorded, and the
mouse was given a rest period of 5 min with access to food and water. This was repeated for a
total of 6 trials. For criteria 3, the mouse would be removed from the study completely. No mice
were removed from the rotarod task. For each mouse, times for the first two trials were removed
so Rotarod data would accurately reflect the mouse's ability to perform the task. The average
time across trials was calculated for each mouse. A two-factor ANOVA with replication was
performed via Microsoft Excel to determine significance.
2.4. Mitochondrial isolation and mitochondrial complex activities
Brain mitochondria was isolated by a Percoll gradient as previously described (Irwin et al.,
2008). The resulting mitochondrial samples were used immediately or stored at −80°C for later
protein and enzymatic assays. During mitochondrial purification, aliquots were collected, and for
confirmation of mitochondrial purity and integrity. Isolated brain mitochondria were plated in
Abcam Mitoscience complex activity assays. Complex I activity was expressed as ODλ450nm as
NADH oxidation (ab109721). Complex II/III was expressed as ODλ550nm as cytochrome c
reduction (ab109905) and complex IV as ODλ550nm as cytochrome c oxidation (ab109911).
69
2.5. Enzyme activities
Transglutaminase activity was measured using the Abcam Transglutaminase Activity Assay
Kit (ab204700) measured colorimetrically at 525 nm. The limit of quantification of this assay is
~10 µU in cell/tissue lysates. Aconitase activity was measured using the Caymen Aconitase
Assay Kit; the reactions were monitored by measuring the increase in absorbance at 340 nm
associated with the formation of NADH proportional to aconitase activity. Pyruvate
Dehydrogenase (PDH) Activity was measured in whole brain homogenate by the abcam PDH
enzyme activity microplate assay (ab109902), which captures fully functional PDH. Activity is
determined by the rate of reduction of NAD
+
to NADH linked to a reporter dye that can be
detected at 450 nm. The phosphofructokinase Activity (PFK) assay kit (abcam155898) measured
PFK1 and PFK2. Their activities were measured in whole brain homogenate and the color
product was detected at 450 nm.
JNK Activity was measured in whole brain homogenate using the abcam JNK1/2
(pT
183
/Y
185
) + Total JNK1/2 elisa kit (abcam176662). Signal is detected at 450nm. Activity is
determined by the ratio of pJNK/JNK. AKT Activity was measured in whole brain homogenate
using the abcam AKT1/2/3 (pS
473
) + AKT Total elisa kit (abcam176657). The signal is detected
at 450 nm. Activity is determined by the ratio of pAKT/AKT.
Nitrate and nitrite concentration – The Cayman Chemical Nitrate/Nitrite Assay Kit measures
total nitrate/nitrite concentration and photometric measurement of the absorbance is due to the
azochromophore corresponding to nitrate concentration. Pyruvate carboxylase (PC)
concentration was measured using the Cloud-Clone Corp. PC immunosorbent assay kit (Product
serial# 6ECC39CD3D). Color was measured at 450 nm. NAD
+
and NADH concentration assay
kit (ab65348) takes measurements of intracellular nucleotides NAD
+
, NADH, and their ratio.
Glutamate concentration was measured in whole brain and isolated brain mitochondria using the
abcam glutamate assay elisa kit (ab83389) that recognizes glutamate as a specific substrate
causing a color development at 450 nm.
70
Mitochondrial biogenesis was expressed as the COXII/ -Globin corresponding to mtDNA
and nDNA. Primers developed by Integrated DNA Technologies. Primer sequences 5’-3’ COXII
F:GCCGACTAAATCAAGCAACA R:CAATGGGCATAAAGC TATGG -Globin; F:GAAG
CGATTCTAGGGAGCAG R:GGAGCAGCGATTCTGAGTAGA. Annealing temp = 56°C.
2.6. Data analysis
Data are reported as means ± SEM of at least three experimental replicates. Group size (n) is
eight unless otherwise stated for specific method. Statistical significance between means was
determined by Student's two-tailed t-test of paired data. The level of statistical significance is
indicated in the respective figures (*p ≤ 0.05, **p ≤ 0.01). Two-way ANOVA between the
sedentary group and exercised group of HD mice was used for rotarod test. Post-hoc analysis
using Student-Neuman Keuls with statistical significance considered if p < 0.05.
3. Results
3.1. Exercise and oxidative mitochondrial metabolism
Cardiovascular exercise leads to the generation of nitric oxide (NO), which is stored as
nitrosothiols and entails Akt activation of eNOS as a major mechanism (Calvert et al., 2011;
Kingwell, 2000; Reid and Durham, 2002; Sessa et al., 2018). Total nitrite and nitrate ([NO2
–
+
NO3
–
]) was measured in brain homogenate of the HD mouse model in both the sedentary group
(483 ± 110 µM) and the exercise group (714 ± 32 µM). Exercise increased NO2
–
+ NO3
–
(surmised as NO) levels in the brain by 48% when compared to the sedentary group (Fig 1A).
NO inhibits transglutaminase cross-linking activity by S-nitrosylation of Cys
277
in the
catalytic core of the enzyme (Jandu et al., 2013). Transglutaminase catalyzes a Ca
2+
-dependent
protein deamidation, transamidation, and cross-linking through addition of the ɛ-amino group of
a lysyl residue to the carboxamide moiety of a glutaminyl residue to form an isopeptide bond
(cross-link) and ammonia (Gorman and Folk, 1980; Nurminskaya and Belkin, 2012).
Transglutaminase activity was elevated in pre-clinical models and post mortem samples of HD
71
patients (Dubinsky, 2017; Jeitner et al., 2001; Kim et al., 2005) and is involved in the
pathogenesis of mitochondrial dysfunction in HD due to the formation of high molecular weight
aggregates of aconitase through cross-linking reactions (Carmo et al., 2018; Kim et al., 2005;
Melino and Piacentini, 1998; Nurminskaya and Belkin, 2012). The majority of transglutaminase-
driven cross-linking occurs in mitochondria (Altuntas et al., 2014; Jandu et al., 2013).
Fig. 1. Effect of treadmill training on metabolic parameters
(A) [NO 2
–
+NO 3
–
] levels were measured in the whole brain homogenate of the HD mouse model. (B)
Transglutaminase activity was measured in isolated brain mitochondria. (C) Aconitase activity was measured in
isolated brain mitochondria. (D) NAD
+
and NADH concentrations. (E) Glutamate levels in mitochondria and whole
brain homogenate. Experimental conditions as described in the Materials and methods section. n =; *p ≤ 0.05.
____________________________________________________________________________________________________________________
72
Transglutaminase activity in isolated brain mitochondria was decreased by 52% in the
exercise group (0.399 ± 0.087 µmol/min/mg protein) as compared to the sedentary group (0.800
± 0.141 µmol/min/mg protein) (Fig 1B). It may be surmised from data in Fig. 1A and from
previous mechanistic studies of transglutaminase crosslinking that the elevated NO levels during
exercise are associated with the decrease in transglutaminase activity. Transglutaminase activity
in whole brain homogenate of the sedentary (0.240 ± 0.024 µmol/min/mg protein) and exercise
(0.184 ± 0.011 µmol/min/mg protein) groups, showed a decrease of ~24%, which did not reach
statistical significance, probably because the effect was diluted by other cellular proteins with the
majority of transglutaminase activity residing within mitochondria (Kim et al., 2005).
Transglutaminase protein levels and activity are highly expressed in liver (Piacentini et al.,
2018). The effects of exercise are systemic, for the transglutaminase activity in liver was also
decreased by exercise (1.241 ± 0.091 µmol/min/mg protein) as compared to the sedentary (2.622
± 0.442 µmol/min/mg protein) group (p < 0.05). These data confirm earlier reports of inhibition
of transglutaminase subjected to a NO flow from a NO-donor (SNAP, S-nitroso-N-
acetylpenicillamine) (Bernassola et al., 1999).
Exercise increased brain mitochondrial aconitase activity by 22%: sedentary group (13.92 ±
0.78 µmol/min/mg protein) compared to the exercise group (17.02 ± 1.05 µmol/min/mg protein)
(Fig 1C). This suggests that the decrease in transglutaminase activity observed in brain and liver
has minimized the crosslinking with aconitase thereby rescuing the latter’s activity. It is unlikely
that peroxynitrite (Schöpfer et al., 2000) or superoxide anion (Hausladen and Fridovich, 1994)
were involved in aconitase inhibition because they elicit an irreversible inactivation of the iron-
sulfur cluster upon loss of labile iron in the enzyme (Han et al., 2005). Thus, the likely
mechanism is that exercise induced an inverse relationship in which aconitase activity is
preserved through NO inactivation of transglutaminase in brain mitochondria.
Aconitase activity is a vital component of the TCA cycle and impacts the production of
reducing equivalents (NADH). Exercise significantly improved aconitase activity in the HD
mouse model (Fig 1C), which impacted downstream levels of reducing equivalents: NAD
+
was
73
increased 6-fold in the exercise group (0.710 ± 0.187 µM) as compared to the sedentary (0.117 ±
0.0729 µM) group (Fig 1D). NADH levels also increased by 22% in the exercise group but did
not reach statistical significance: sedentary (1.918 ± 0.297 µM) versus exercise (2.330 ± 0.051
µM) (Fig 1D). The significant increase in NAD
+
suggests NADH utilization at a high rate due to
enhanced mitochondrial complex activities (Fig 2A-C).
The neurotransmitter glutamate is generated from α-Ketoglutarate in the TCA cycle, and its
impaired production and transport has been documented in Huntington’s disease (Rebec, 2018).
Glutamate levels measured in whole brain homogenate and in isolated brain mitochondria
showed increases of 60% (sedentary (17.7 ± 4.9 µM) versus exercise (28.4 ± 4.9 µM)) and 68%
(sedentary (2.25 ± 0.25 µM) versus exercise (3.78 ± 1.07 µM)) (Fig 1E), respectively, albeit they
did not reach statistical significance.
74
Fig. 2. Effect of treadmill training on mitochondrial electron transfer complexes and mitochondrial biogenesis
(A) Complex I; (B) Complex II/III; (C) Complex IV; (D) mitochondrial biogenesis expressed as mtDNA/nDNA
values. Experimental conditions as described in the Materials and methods section. n =; *p ≤ 0.05, **p ≤ 0.01.
____________________________________________________________________________________________________________________
Accordingly, the increase in NAD
+
levels (Fig 1D) elicited by exercise were linked to
increased mitochondrial complex activity relative to sedentary animals; complex I (41%) (Fig
2A) (sedentary (1.93 ± 0.14 mOD/min/mg protein) versus exercise (2.725 ± 0.20 mOD/min/ mg
protein)), complex II/III (31%) (Fig 2B) (sedentary (1.24 ± 0.25 mOD/min/ mg protein) versus
exercise (1.62 ± 0.24 mOD/min/ mg protein)), and complex IV (58%) (Fig 2C) (sedentary (1.11
± 0.07 mOD/min/ mg protein) versus exercise (1.77 ± 0.23 mOD/min/ mg protein)). The
3
2
0
Complex I
(mOD/min/mg protein)
Sedentary Exercise
A
**
1
2
0
Complex II/III
(mOD/min/mg protein)
Sedentary Exercise
B
*
1
2
0
Complex IV
(mOD/min/mg protein)
Sedentary Exercise
C
**
1
4
0
mtDNA/nDNA x 10
–4
Sedentary Exercise
D
2
75
increased flow of reducing equivalents across the complexes suggests higher rate of energy
production in the form of proton gradient generated ATP.
Fig. 3. Glucose and pyruvate metabolism
(A) PFK1/2 activity; (B) Pyruvate dehydrogenase (PDH) activity. (C) Pyruvate carboxylase content. Experimental
conditions as described in the Materials and methods section. n =; *p ≤ 0.05, **p ≤ 0.01.
___________________________________________________________________________________________________________________
Mitochondrial biogenesis cannot account for the increased complex activities because the
mtDNA/nDNA values were not affected by exercise (Fig 2D): sedentary (2.20 ± 0.573 x 10
-4
)
versus exercise (3.03 ± 0.815 x 10
-4
). Several deficiencies of mitochondrial complex activities
within the electron transport chain have been observed in preclinical and clinical HD (Browne et
al., 1997; Carmo et al., 2018; Gu et al., 1996; Johri et al., 2013).
Phosphofructokinase (PFK) is the key regulator of glycolysis leading to pyruvate formation
in cytosol and its further mitochondrial metabolism upon oxidative decarboxylation by the
pyruvate dehydrogenase complex. Total phosphofructokinase activity (combination of PFK1 and
PFK2, converting fructose-6-phosphate to fructose-(1/2),6-diphosphate) was increased by 17%
in the exercise group (3.486 ± 0.131 µmol/min/mg protein) as compared with sedentary (2.98 ±
0.135 µmol/min/mg protein) (Fig 3A). This might be consistent with previous reports on NO
augmenting glycolysis in astrocytes through activation of PFK2 (Almeida et al., 2004). The
pyruvate dehydrogenase (PDH) complex converts pyruvate to acetyl-CoA through oxidative
4
0
Pyruvate dehydrogenase
( µmol/min/mg protein)
Sedentary Exercise
B
**
2
4
0
PhosphoFructoKinase 1/2
( µmol/min/mg protein)
Sedentary Exercise
A
*
2
5.0
0
Pyruvate carboxylase
(ng/mg protein)
Sedentary Exercise
C
*
2.5
76
decarboxylation; the complex activity was increased by 18% in the exercise (3.68 ± 0.104
µmol/min/mg protein) group versus sedentary (3.12 ± 0.227 µmol/min/mg protein) group (Fig
3B). Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate, the acceptor of acetyl-CoA in
the TCA. Pyruvate carboxylase is expressed in astrocytes, and astrocytes are the sole de novo
synthesizers of glutamate from glucose in the CNS (Verkhratsky and Nedergaard, 2018).
Exercise increased pyruvate carboxylase concentration by 64% (4.16 ± 0.23 ng/mg protein)
compared to sedentary (2.53 ±0.19 ng/mg protein) (Fig 3C).
3.2. Exercise and insulin signaling
Insulin signaling plays a pivotal role in not only peripheral tissues, but also the central
nervous system where it participates in neuronal survival, synaptic plasticity, memory and
learning (Craft and Watson, 2004; van der Heide et al., 2006). Neuronal energy deficits are
implicated in several neurodegenerative disorders including Huntington’s disease (Liu et al.,
2008; Mochel et al., 2007).
Fig. 4. Treadmill training on JNK and Akt activation
(A) Akt activation (expressed as pAkt/Akt); (B) pJNK/JNK.
Experimental conditions as described in the Materials and methods
section. n =; *p ≤ 0.05.
_______________________________________________________________________________________
0.06
0
pAkt/Akt
Sedentary Exercise
A
*
0.03
3
0
pJNK/JNK
Sedentary Exercise
B
*
2
1
77
Insulin signaling is regulated by the stress-responsive Jun-N-terminal kinase (JNK) and
both signaling processes interact to regulate metabolic homeostasis. Akt activity was increased
by 22% in the exercise (pAkt/Akt = 0.055 ± 0.0016) group as compared to the sedentary
(pAkt/Akt = 0.045 ± 0.0032) group (Fig 4A). JNK activity was decreased by 27% in the exercise
(pJNK/JNK = 2.13 ± 0.19) group when compared with the sedentary (pJNK/JNK = 2.93 ± 0.24)
group (Fig 4B). These data may confirm previous reports that endogenous NO negatively
regulates JNK activation by S-nitrosylation (Park et al., 2000).
3.3. Exercise and Physical and Cognitive Performance
These improvements in brain metabolism due to chronic exercise intervention build a
mechanistic platform for the significant improvement in behavioral outcome seen in HD mice.
Previous work has shown the rotarod test to be an accurate measure for motor function and skill
based learning in animal models (Deacon, 2013) (Shiotsuki et al., 2010; Stefanko et al., 2017;
Wang et al., 2016 ). The rotarod challenges mice both physically to stay on the rod along with
cognitively to anticipate change in speed and direction. The motor behavior in HD mice was
evaluated on the accelerated rotarod using latency to fall (expressed as s). Exercise (37.0 ± 8.6 s)
significantly improved latency to fall in HD mice by 73% as compared to the sedentary (21.3 ±
3.5 s) group (Fig 5).
Fig. 5. Skill-based learning as assessed by rotarod test
Rotarod latency to fall, expressed in s. Behavioral testing as described in the
Materials and methods section. n =; *p ≤ 0.05.
50
0
Rotarod Latency to Fall
(s)
Sedentary
Exercise
*
25
78
4. Discussion
This study demonstrates that the intervention strategy of chronic cardiovascular exercise by
treadmill training leads to an improved functional outcome as assessed by the rotarod latency to
fall (Fig 5). The mechanistic platform for this functional outcome of skill-based learning and
motor coordination may be based on changes of the HD metabolic phenotype. Data from this
study are summarized in Fig. 6.
Transglutaminase activity is elevated while aconitase activity is decreased in HD (Karpuj et
al., 1999; Lesort et al., 1999; Tabrizi et al., 1999). Although a complete understanding of why
transglutaminase activity is elevated in HD and other neurodegenerative diseases is unknown, a
79
Fig. 6. Effects of treadmill training on metabolic parameters
The figure summarizes the findings in this study. Positive effects of treadmill training (resulting in brain
[NO 2
–
+NO 3
–
] are indicated by a + arrow. Inhibition by a – arrow. MPC, mitochondrial pyruvate carrier;
MCT, monocarboxylate transporter; PFK1/2, phosphofructokinase 1 and 2. PDH, pyruvate
dehydrogenase; SCOT, succinyl-CoA transferase; TG, transglutaminase.
glucose
glucose
pyruvate
pyruvate
GLUT
ketone
bodies
MCT
oxalo-
acetate
citrate
isocitrate
malate
NADH
oxoglutarate
succinyl-CoA
fumarate
succinate
SCOT
acetyl-CoA
MPC
glycolysis
I III IV Q
c
V
glutamate
ADP
ATP
SYNAPTIC
PLASTICITY
GDH
PDH
PC
MCT
aconitase
KGDH
TG NO
Exercise
(Treadmill)
Functional Outcome
(Rotarod)
aconitase-X
PFK1
ketone
bodies
NAD
+
80
likely combination of impacting features was advanced. (Citron et al., 2002; Johnson et al., 1997;
Kim et al., 1999; Kim et al., 2005). Transglutaminase may be assisting with the generation of
senile plaques, nuclear inclusions, and Lewy bodies causing cytotoxicity; additionally,
transglutaminase may sensitize cells to apoptosis (de Cristofaro et al., 1999; Piacentini et al.,
2002). Evidence for a role of transglutaminase in HD originates from a transglutaminase
knockout R6/1 HD mouse model that reported reduction in neuronal death, improved behavior,
and prolonged survival (Mastroberardino et al., 2002). In this study decreases were observed in
transglutaminase activity in brain mitochondria in the exercised HD mouse model. A systemic
effect elicited by chronic cardiovascular exercise is suggested by the observed decrease of
transglutaminase activity in liver, but confirmation would depend on the transglutaminase
content and activity of specific tissues.
The aberrant activity of transglutaminase in HD is associated with mitochondrial
dysfunction, focusing on aconitase inactivation by cross-linking (Kim et al., 2005). Currently, it
is unclear whether aconitase acts as an acyl acceptor, donor, or both, i.e., which mitochondrial
proteins aconitase is bound to and whether aconitase could be bound to itself. However,
regardless of the identity of the second substrate, it is clear that transglutaminase inactivates
aconitase in HD. NO inactivates transglutaminase by disrupting its catalytic core: the increase of
brain [NO2
–
+ NO3
–
] (Fig 1A) following treadmill training supports the notion that the observed
decrease in brain mitochondrial transglutaminase activity (Fig 1B) is a consequence of its
inhibition by NO, thus preventing its crosslinking inactivation of aconitase (Fig 1C).
The preservation of aconitase activity further improves the generation of reducing
equivalents in the TCA cycle. NAD
+
was significantly increased while NADH was not in the
exercised HD mice (Fig 1D), which was interpreted as the kinetic control exerted by the high
electron flow through the respiratory complexes I, II/III, and IV (Fig 2A-C). Although
deficiencies of all mitochondrial complexes have been reported in preclinical and clinical HD,
the majority of these are seen in complex II and III (Browne et al., 1997; Carmo et al., 2018;
Dubinsky, 2017; Gu et al., 1996; Johri et al., 2013). However, the effect of exercise seems to
81
affect complexes I-IV of the respiratory chain (Fig 2A-C). The neurotransmitter glutamate has
been associated with the metabolic dysfunction in HD (Rebec, 2018), albeit the substantial
increase elicited by exercise was not statistically significant. Supporting a high formation of
reducing equivalents in the TCA cycle are (a) faster glycolysis by conversion of glucose to
pyruvate upon increased PFK1/2 activity (Fig 3A); (b) delivery of acetyl-CoA to the cycle upon
increased PDH activity (Fig 3B); and (c) are anaplerotic mechanism sustained by way of
pyruvate carboxylase levels (Fig 3C). PFK and PDH are impaired in HD human brain in caudate,
putamen, and globus pallidus and R6/2 striatum as well as glucose uptake and GLUT transporter
expression (Dubinsky, 2017).
Cardiovascular exercise impacts positively glucose transporter expression and insulin
signaling through phosphorylation of Akt in skeletal muscle (Arias et al., 2007; Bruss et al.,
2005; Richter and Hargreaves, 2013). The PI3K/AKT route of insulin signaling and MAPK
stress-sensitive JNK regulate energy metabolism: activation of both signaling pathways are
affected by chronic exercise (Fig 4A-B). Likewise, the significance of Akt/JNK homeostasis in
brain aging and Huntington’s disease has been documented (Colin et al., 2005; Jiang et al., 2013;
Yin et al., 2014). Akt insulin signaling facilitates the translocation of GLUT 3/4 in brain to the
plasma membrane, promotes glycolysis, and enhances mitochondrial function through Akt
translocation into mitochondria (Yin et al., 2016a).
This metabolic platform entailing NO-derived inhibition of transglutaminase activity and
concomitant rescue of aconitase activity, increase in glycolysis and mitochondrial oxidative
metabolism, and the signaling pathways that impinge and modulate energy metabolism are
depicted in Fig. 6. These pathways attempt to provide mechanistic insights between the initial
input (treadmill training) and the functional outcome (rotarod) mediated by the increase in brain
[NO2
–
+ NO3
–
] elicited by the former. An association with rotarod performance and T-maze
improvement in the CAG140KI mouse model (Stefanko et al., 2017) strengthens the skill-based
learning by the rotarod approach.
82
Acknowledgements – This project was made possible by the financial support from the
Packer/Wenz Endowment. I’d like to acknowledge and thank my PI Dr. Enrique Cadenas and
two the committee members Dr. Michael Jakowec and Giselle Petzinger for their guidance on
this project. The important help from two other students who assisted in some of the sacing,
behavior tests, and mitochondrial isolation: Alicia Warnecke and Elliott Cheung.
83
CHAPTER 5
CONCLUDING REMARKS
Over the course of my 5 years here at USC School of Pharmacy I have gained a deeper
understanding of the aging brain and neurodegenerative disease in the context of metabolism.
Earliest work focused on mitochondrial function and respiratory efficiency. Learning under Dr.
Brinton with the help of Dr. Jia Yao and Dr. Ron Irwin I developed a base understanding for the
impact of brain mitochondria in Alzheimer’s disease. Though the large 3xTgAD mouse study
was not mechanistic in nature it combined biochemical markers along with cognitive behavioral
outcomes to determine a therapeutic effect. The PhytoSERM project structure was very complex
and therefore leaves many conclusions about the formulation efficacy unknown. However, it did
teach the important of sex in preclinical studies. Over the course of the yearlong chronic
prevention study, I observed that male and female 3xTgAD mice had significant differences in
the non-treatment group and in their responses to treatment. Beyond expected differences in
weight and feeding habits they had significant differences in amyloid pathological load in the
brain. Significant differences also resided in several metabolic markers in the blood. Treated
3xTgAD mice had sex specific responses in behavioral outcomes and in brain mitochondrial
respiration. This shows the importance of studying sex response in models that have differences
or haven’t been studied before. Future of neurotherapeutic development is in personalized
medicine. This will require deeper research in to differing patient characteristics beyond sex.
Genetic backgrounds, epidemiological factors, comorbidities, diet, exercise, cognitive training,
may all play key roles in improving therapeutic efficacy and understanding neurodegenerative
diseases.
An addental outcome from the PhytoSERM study was the lack in change in amyloid beta
42, a previously suspected hallmark of Alzheimer’s cognitive deficiency, and a significant
improvement in cognitive behavioral performance in the novel object recognition test. This
agrees with the recent transition in Alzheimer’s therapeutic strategy targets switching from
84
previous beliefs of amyloid as a causative factor to more mechanistic metabolic and genetic
factors prior to the plaques formation.
I was exposed to a new strategy for neurodegenerative disease intervention, exercise,
during my clinical rotation with Dr. Petzinger and my preclinical exercise work with Dr.
Cadenas and Dr. Jakowec. In the clinic I witnessed the direct benefits cardiovascular exercise
and differing environmental stimulation had on Parkinson’s patients’ disease progression. In the
lab using the CAG140KI mouse model for Huntington’s I explored possible mechanisms for the
improvement from exercise. The combination of reading previous research and discussions of
brain metabolism with my PI Dr. Cadenas lead me to focus on the elevation of nitric oxide to be
a key factor in the function outcome improvements from chronic cardiovascular exercise. Further
investigation linked this NO elevation to an inactivation of an enzyme involved with a
crosslinking reaction reducing metabolic dysfunction and neuronal damaging protein aggregates.
Exercise also had influence over insulin resistance pathways and glucose metabolism. In the is
HD study I was able to show that the functional outcome of this study was backed by several
metabolic changes due to the exercise intervention.
The HD study differed from the AD study greatly. It taught me the importance of
mechanistic research and asking deeper questions on how interventions interact with the diseases
state. All three of my committee members were vital in helping challenge me to grow and
become a better scientist. Ask more questions, dig deeper into how mechanistic pathways
interact with each other to produce a behavioral outcome. I cannot thank them enough for this
experience.
Though my immediate future career is not as a postdoctoral scientist the things I have
learned over the 5 years and the experiences I’ve had has well prepared me for my next step. I
will be working as a Strategic Insights and Planning associate consultant at ZS Associates in
their business consulting division of biopharmaceuticals out in their thousand oaks office.
Consulting is all about problem solving and using research findings to create better outcomes for
85
therapeutic developers and patients alike. Business consulting in biopharma and healthcare
requires the ability to build and defend arguments backed by evidence. The experience of writing
my HD exercise paper, defending my qualifying exam and many other presentation opportunities
throughout the last 5 years have well prepared me for this career.
86
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Abstract (if available)
Abstract
This thesis is a collection of work by Charles C. Caldwell during his doctoral studies at University of Southern California School of Pharmacy in the major of Clinical and experimental therapeutics. Topics of focus include brain aging and neurodegenerative disease, sex differences in animal models and therapeutic response, clinical outcomes in comparison with preclinical investigations, and exercise as a viable modulator of brain energy metabolism. Neurodegenerative diseases in this thesis include Alzheimer’s, Parkinson’s, and Huntington’s. Drug and non-drug-based interventions impacted glycolytic and mitochondrial metabolic function resulting in cognitive and motor functional improvements.
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Asset Metadata
Creator
Caldwell, Charles Clarke
(author)
Core Title
Exploration and expansion of novel therapeutic strategies targeting brain metabolism in neurodegenerative diseases
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Clinical and Experimental Therapeutics
Publication Date
11/21/2018
Defense Date
10/23/2018
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Alzheimer's,brain metabolism,Exercise,Huntington's,OAI-PMH Harvest,Parkinson's,phytoSERMs,therapeutics
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Cadenas, Enrique (
committee chair
), Jakowec, Michael (
committee member
), Petzinger, Giselle (
committee member
)
Creator Email
cccaldwe@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-108082
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UC11675190
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etd-CaldwellCh-6979.pdf (filename),usctheses-c89-108082 (legacy record id)
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etd-CaldwellCh-6979.pdf
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108082
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Dissertation
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Caldwell, Charles Clarke
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University of Southern California Dissertations and Theses
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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 a...
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Tags
Alzheimer's
brain metabolism
Huntington's
Parkinson's
phytoSERMs
therapeutics