Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Monoamine oxidase (MAO) knock-out mouse models: Tools for studying the molecular basis of aggression, anxiety, autism and cancers
(USC Thesis Other)
Monoamine oxidase (MAO) knock-out mouse models: Tools for studying the molecular basis of aggression, anxiety, autism and cancers
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Copyright 2024 Zijin Gao
Monoamine oxidase (MAO) knock-out mouse
models: Tools for studying the molecular basis of
aggression, anxiety, autism and cancers
by
Zijin Gao
A Thesis Presented to the
FACULTY OF THE USC MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2024
ii
Acknowledgements
I would like to express my deepest appreciation to Dr. Jean C. Shih and everyone in the Shih
Lab for all the provided knowledge and expertise, patience, and support. I’m also extremely
grateful to my thesis committee Dr. Yong Zhang and Dr. Angel P. Tabancay Jr. Thanks for all the
valuable suggestions and feedbacks.
I’d also wish to thank my family for their love and support.
A special thanks to Vy, Rachel, and Emily for the company during my writing.
iii
Table of Contents
Acknowledgements....................................................................................................................... ii
List of Tables................................................................................................................................ iv
List of Figures ............................................................................................................................... v
Abbreviations............................................................................................................................... vi
Abstract ....................................................................................................................................... vii
Introduction ................................................................................................................................... 1
Chapter 1. MAO A-KO mice show aggression and MAO A/B-KO mice show aggression
and anxiety behaviors.................................................................................................................... 5
1.1 Generation of MAO A-KO mouse, alteration of substrates, and aggressive
behavior....................................................................................................................... 5
1.2 Generation of MAO B-KO mouse, alteration of substrates, and reduced
anxiety ......................................................................................................................... 9
1.3 Generation of MAO A/B-KO mouse, alteration of substrates, aggression,
and anxiety behavior ................................................................................................. 11
Chapter 2. MAO A-KO mouse and MAO A/B-KO mouse show symptoms of autism
spectrum disorder........................................................................................................................ 14
2.1 MAO A-KO mouse exhibits ASD features in behavioral test and
neuropathology analysis............................................................................................ 14
2.2 MAO A/B-KO mouse exhibits ASD features in behavioral test and
neuropathology analysis............................................................................................ 17
Chapter 3. MAO A-KO mouse shows suppressed growth of prostate cancer and
melanoma .................................................................................................................................... 19
3.1 MAO A-KO cells reduce ROS production and reverse EMT in prostate
cancer ........................................................................................................................ 19
3.2 MAO A-KO mouse show inhibited TAM polarization in melanoma................. 23
Chapter 4. Discussion.................................................................................................................. 26
4.1 The role of MAO in neurotransmitter metabolism, aggression, and anxiety...... 26
4.2 The role of MAO A in regulating behaviors and brain structures
associated with autism spectrum disorder................................................................. 28
4.3 MAO A and potential therapeutic implications in neuroblastoma...................... 29
Bibliography................................................................................................................................ 33
iv
List of Tables
Table 1. The relative levels of neurotransmitters and their substrates in the brain of MAO
knockout mice compared to wild-type.................................................................................. 12
Table 2. The behavioral test results of MAO KO mice compared to wild-type on
aggression, anxiety, and depression ...................................................................................... 13
Table 3. The behavioral test results of MAO KO mice compared to wild-type on autism
spectrum disorder.................................................................................................................. 17
v
List of Figures
Figure 1. MAO A and MAO B amino acid sequence alignment .................................................... 2
Figure 2. MAO catalyzed reactions. ............................................................................................... 3
Figure 3. Reactive oxygen species (ROS) triggering Epithelial-to-mesenchymal transition
pathway................................................................................................................................. 21
vi
Abbreviations
Abbreviation Definition
3-MT 3-methoxytyramine
5-HIAA 2-(5-hydroxy-1H-indol-3-yl) acetic acid
5-HIAL 2-(5-hydroxy-1H-indol-3-yl) acetaldehyde
5-HT 5-hydroxytryptamine; serotonin
ADHD Attention-deficit/hyperactivity disorder
ALDH aldehyde hydrogenase
AR aldose reductase
ASD Autism spectrum disorder
ASP Antisocial personality disorder
COMT catechol-O-methyltransferase
DA Dopamine
DHPG 4-(1,2-dihydroxyethyl) benzene-1,2-diol
DOMA 2-(3,4-dihydroxyphenyl)-2-hydroxyacetic
acid
DOPAC 2-(3,4-dihydroxyphenyl) acetic acid
DOPAL 2-(3,4-dihydroxyphenyl) acetaldehyde
DOPGAL 2-(3,4-dihydroxyphenyl)-2-
hydroxyacetaldehyde
EMT Epithelial-to-mesenchymal transition
EPM Elevated plus-maze test
FST Forced swim test
HIF- 1α Hypoxia-inducible factor 1 alpha
HVA 2-(4-hydroxy-3-methoxyphenyl) acetic acid
KO Knockout
MAO Monoamine oxidase
MHPG 1-(3-hydroxy-4-methoxyphenyl) ethane-1,2-
diol
NE norepinephrine
NHEJ Non-homologous end-joining
PCa Prostate cancer
PEA Phenylethylamine
PFC Prefrontal cortex
PHD Prolyl hydroxylase domain
RI test Resident-intruder test
ROS Reactive oxygen species
TAM Tumor associated macrophage
TME Tumor microenvironment
VHL Von Hippel-Lindau
vii
Abstract
Monoamine oxidase (MAO) is an enzyme involved in the metabolism of neurotransmitters
such as serotonin (5-HIAA), norepinephrine (NE), dopamine (DA), and phenylethylamine
(PEA). In this paper, we discuss research using MAO knockout (KO) mice that provide insights
into various conditions. Studies by Olivier Cases (Cases et al. 1995) showed that MAO A
knockout (KO) mouse models exhibit significantly elevated levels of serotonin, dopamine, and
norepinephrine, leading to increased aggressive behavior. In contrast, MAO B KO mice, with
significantly elevated phenylethylamine levels, did not display aggressive behavior but exhibit
less anxiety and depressive-like behaviors in studies by Joseph Grimsby et al (Grimsby et al.
1991). Studies by Kevin Chen (Chen et al. 2004) revealed that MAO A/B-KO mice show
heightened anxiety-like behavior. Study by Marco Bortolato et al. (Bortolato et al. 2008) showed
that MAO A-KO and MAO A/B-KO mice exhibit pronounced features of autism spectrum
disorder (ASD). Jason Boyang Wu's study on prostate cancer (Wu et al. 2014) demonstrated that
MAO A promotes cancer progression through HIF-1α stabilization and epithelial-tomesenchymal transition (EMT), suggesting MAO A as a therapeutic target. Yu-Chen Wang's
research on melanoma (Wang et al. 2021) highlighted MAO A's role in regulating tumorassociated macrophage (TAMs) polarization, indicating that targeting MAO A could modulate
the tumor microenvironment and improve cancer treatment outcomes. In this paper, we discuss
the multifaceted role of MAO enzyme in neurobiological and oncological processes, providing
us with clues of possible treatment strategies and research directions that can be applied to other
diseases.
1
Introduction
Monoamine oxidase A (MAO A) and B (MAO B) are two isoenzymes crucial for the
breakdown of monoamine neurotransmitters, including serotonin (5-hydroxytryptophan, 5-HT),
dopamine (3,4-dihydroxyphenethylamine, DA), phenylethylamine (PEA), and epinephrine,
which are vital for regulating sleep, appetite, mood, and stress (Medline Genetics 2017; Shih
2018).
The MAO A and MAO B genes are located on the Xp11.23-Xp22.1 region of the X
chromosome but are oriented in opposite directions (Lan et al., 1998; Murphy 2006). The MAO
A and MAO B are called isoenzymes because they are encoded by different genes with 70%
amino acids identical (Bach et al. 1988) (Figure 1). Both genes contain 15 exons and 14 introns,
with all the introns interrupting the coding sequence at the same position. They exhibit identical
exon-intron organization, suggesting a common ancestor gene origin (Grimsby et al. 1991). The
studies by Bach discovered the amino acid sequences of MAO A and MAO B in humans (Bach
et al. 1988) and rodent animals (Ito et al. 1988; Kuwahara et al. 1990), allowing the generation of
the MAO-KO mice, which further helped with the study of the molecular basis of monoamine
and potential drug development.
2
Figure 1. MAO A and MAO B amino acid sequence alignment. Data obtained from NCBI 2004. Matrix: EBLOSUM62; Gap
penalty: 2.0; Extend penalty: 2.0; Score: 2082.0; MAO A sequence length: 527; MAO B sequence length: 520; Alignment length:
536; Identity: 381/536 (71.08%); Similarity: 454/536 (84.70%); Gaps: 25/536 (4.66%).
The abnormal level of MAO activity has been shown linked to various psychiatric disorders.
MAO A deficiency was first identified in a Dutch family, where 14 males exhibited a C936T
mutation in exon 8 of the MAO A gene, resulting in no detectable MAO A expression on
Northern blot analysis. This condition is known as Brunner Syndrome (Brunner et al. 1993). As
Brunner syndrome is an X-linked recessive disorder (Brunner et al. 1993), MAO A deficiency
occurs exclusively in males and is characterized by a diminished ability to control impulses,
leading to aggressive behaviors and violent outbursts. Affected individuals may also experience
intellectual disabilities and difficulties with social interactions. MAO A deficiency is often
associated with other neurological disorders, such as autism spectrum disorder (ASD), attentiondeficit/hyperactivity disorder (ADHD), and antisocial personality disorder (ASP) (Shih, 2018).
In contrast, MAO B deficiency does not present any symptoms (Chen et al. 2004).
3
Figure 2. MAO catalyzed reactions. Adapted from Obata et al. 2022. Substrates: 5-HIAL: 2-(5-hydroxy-1H-indol-3-
yl)acetaldehyde; 5-HIAA: 2-(5-hydroxy-1H-indol-3-yl)acetic acid; DOPAL: 2-(3,4-dihydroxyphenyl)acetaldehyde; DOPAC: 2-
(3,4-dihydroxyphenyl)acetic acid; HVA: 2-(4-hydroxy-3-methoxyphenyl)acetic acid; DOPGAL: 2-(3,4-dihydroxyphenyl)-2-
hydroxyacetaldehyde; DOMA: 2-(3,4-dihydroxyphenyl)-2-hydroxyacetic acid; DHPG: 4-(1,2-dihydroxyethyl)benzene-1,2-diol;
MHPG: 1-(3-hydroxy-4-methoxyphenyl)ethane-1,2-diol. Enzymes: ALDH: aldehyde hydrogenase; AR: aldose reductase; COMT:
catechol-O-methyltransferase.
As an MAO substrate, serotonin primarily affects the prefrontal cortex (PFC), particularly the
orbitomedial and ventromedial regions, which are responsible for regulating negative emotions
and anger (Seo et al. 2008). Increased serotonin levels in the PFC enhance inhibitory synaptic
transmission, thereby reducing impulsivity. Previous studies have shown that low levels of 5-
HIAA, a serotonin metabolite (Figure 2), are associated with aggression, violent suicide
attempts, impulsive murder, and recidivism in murderers (Soubrié 1986).
An impaired serotonin system can also lead to hyperactivity of the dopamine system. Its
interaction with dopamine also contributes to impulse regulation. The dopamine level rises in
aggressive fights, and serotonin and dopamine concentrations are negatively associated (Seo et
4
al. 2008). The neuronal cell bodies and terminal sites of dopamine neurons are modulated by
serotonin. Activation of serotonin receptor 5-HT2 inhibits dopamine activity, while 5-HT2
receptor antagonists could stop this inhibition. The suppressed serotonin system may result in a
hyperactive dopamine system, promoting impulsive behavior.
Previous studies have shown that serotonin, dopamine, and other monoamine
neurotransmitters play a role in aggression, emotion regulation, and ASD. As the monoamine
metabolizing enzyme, it can be hypothesized that MAO is associated with these diseases and
may be utilized as a research target. MAO A, MAO B, and MAO A/B gene knockout (KO)
mouse models were applied in various studies to help us understand the in vivo function of the
isoenzymes, providing insight into possible drug treatments.
The MAO-catalyzed reaction also produces reactive oxygen species (ROS). When
monoamine is oxidized into aldehyde, water is involved in this process and oxidized into
hydrogen peroxide (Segura-Aguilar et al. 2014). As a byproduct of monoamine degradation,
H2O2 is then further reacted with ferrous ions to generate hydroxyl radicals (HO.), which belong
to the group of compounds called reactive oxygen species (Drechsel & Patel 2008). These ROS
can cause cellular damage, contributing to the initiation and progression of tumors. Using MAO
A, MAO B, or MAO A/B double KO mice, allows us to study the production of ROS and the
development of diseases such as cancer.
5
Chapter 1. MAO A-KO mice show aggression and MAO A/B-KO
mice show aggression and anxiety behaviors
Anxiety and aggression are critical areas of study not only in neurobiology but also in
sociology due to their significant impact on both individual behavior and social dynamics.
Anxiety is an emotional response to stressful and threatening situations. While moderate levels
of anxiety and fear can enhance an individual's potential and preparedness, excessive anxiety can
become pathological, leading to conditions such as panic attacks and social anxiety disorder
(Neumann 2010). On the other hand, aggression serves essential purposes such as declaring
ownership of territory, securing mating opportunities, and acquiring food in the nature world
(Neumann 2010). When the aggression is excessively expressed, which often stems from
deficient emotion regulation and disrupted neuroendocrine functioning, impaired social
behaviors such as violence occur, which would hinder social interaction, making it challenging
for affected individuals to form and maintain healthy relationships and engage in socially
appropriate behaviors (Neumann 2010).
As serotonin and dopamine are important regulators of emotion and impulse, the relationship
between MAO-KO mice, serotonin and dopamine concentration, and aggressive or anxiety
behaviors were tested.
1.1 Generation of MAO A-KO mouse, alteration of substrates, and aggressive
behavior
MAO A-KO mouse is achieved through inserting an interferon-B gene into the MAO A gene
in C3H/HeJ mice that causes the deletion of exon 2 and 3 of the MAO A gene. The mouse then
6
expresses no MAO A activity in the brain and liver. These mice are known as Tg8 mice, which
are commonly used as MAO A-KO mouse models in experiments (Cases et al. 1995).
Compared to wild-type mice, MAO A knockout mice showed altered neurotransmitter levels,
including increased concentrations of serotonin, dopamine, and norepinephrine (NE), and a
decreased level of 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite (Chen et al.
2004) (Table 1). This suggests that serotonin, dopamine, and norepinephrine are the preferred
substrates for MAO A.
Behavior changes were observed as well. MAO A-KO mice exhibited significantly reduced
body weight, increased aggressive behavior, shorter latency to attack, social-communicative
deficits, and over-generalized fear conditioning. These behavior changes were further tested and
quantified.
In animal models, aggression primarily functions as a means to threaten opponents in defense
of territory and resources, rather than to inflict physical harm. For rodents, the expression of
offensive aggression typically involves behaviors such as piloerection, which makes them appear
larger and more intimidating, and lateral threat displays, where they arch their backs and expose
their flanks to scare off intruders. Defensive aggression, characterized by sudden attacks on
vulnerable body parts without prior signaling, is reserved for life-threatening situations
(Neumann 2010). Therefore, aggressive behaviors involving attacks on vulnerable body parts
under non-life-threatening conditions are considered abnormal and may indicate high levels of
stress or genetic predispositions. One of the most common tests for assessing aggression in
rodent models is the resident-intruder (RI) test (Neumann 2010). In this test, an experimental
7
male rat is initially housed with a female rat for several days to trigger territorial aggression.
After this period, the female rat is replaced with an unfamiliar male rat, allowing the
experimental rat to exhibit offensive aggression within its home cage. Researchers measure
various parameters such as the latency to the first attack, the frequency of attacks, and the
duration of each attack to quantify aggressive behavior.
In this study by Vishnivetskaya, the predatory behavior test, specifically cricket killing, and
multiple resident-intruder tests, which aim at assessing aggression and social behaviors, were
examined in MAO A deficient mice (Table 2). The C3H/HeJ mice served as the wild-type control
group, while the Tg8 mice represented the MAO A knockout (MAO A-KO) group
(Vishnivetskaya et al. 2007).
In the cricket killing test, which observes the reaction of the mice to the cricket when they
were kept in no food or water condition, both Tg8 mice and C3H mice showed a similar
percentage of predating crickets behavior, yet Tg8 mice exhibited a notably shorter latency to
initiate their attacks. When confronted with BALB/c intruders, Tg8 mice demonstrated reduced
exploration behavior compared to wild-type mice. They also had a higher percentage of attack
(80%-100%) than wild-type (50%-66%). Conversely, while wild-type C3H mice remained
passive in the presence of non-aggressive A/Sn strain intruders, Tg8 mice displayed aggression
toward all intruders, regardless of whether they were the non-aggressive A/Sn mice or mildaggressive BALD/c mice. When presented with an anesthetized intruder, Tg8 mice exhibited
decreased social interaction, such as sniffing the intruder’s body and tail, and showed no
aggression towards the immobile intruder. Similarly, when encountering juvenile intruders, Tg8
8
mice displayed diminished social interaction and occasional aggression, although not
significantly different from wild-type mice. When the position of resident and intruder reverses,
which means when Tg8 mice were put in the cage as intruders while C3H mice as the residents,
60% of Tg8 mice displayed aggressive behavior towards C3H residents, and another 10%
presented threatening postures. It is a notably higher percentage of attacking compared to wildtype intruders (10%). A shorter latency to the first attack of 378.3±771.6 seconds was observed
as well. Moreover, social isolation, in which each resident was isolated in individual housing for
6 weeks prior to the resident-intruder test significantly decreased the latency to the first attack in
Tg8 mice. The percentage of Tg8 mice attacking the intruder during the first 20 seconds of the
test went up to 80%, which is greatly increased compared to the 20% in group with no social
isolation. All 100% of the Tg8 mice attacked the intruder during the first 60 sec of the test, while
only two out of eight C3H males, which is 25%, did so after prolonged social isolation.
For quantifying anxiety in rodent models, tests such as the open field test and the elevated
plus-maze (EPM) test are typically accessed (Neumann 2010). These tests exploit the natural
conflict rodents experience between their tendency to avoid lighted and open areas and their
instinct to explore outside environments. The extent to which rodents avoid open areas in these
tests is used as an indicator of their anxiety levels, with greater avoidance behavior correlating
with higher anxiety.
Studies by Chen revealed that MAO A-KO mice show fewer exploration attempts and shorter
stays in open areas. This may indicate a higher level of anxiety when MAO A enzyme is not
present. However, MAO A knockout mice showed no significantly different behavior from wild-
9
type mice in EPM test.
In conclusion, MAO A-KO mice have higher serotonin, dopamine, and norepinephrine
levels. They show no difference from wild-type when facing immobile intruders or juvenile
intruders in the RI test. However, when facing intruders with similar ages and body sizes, no
matter how aggressive the intruder is, MAO A-KO mice would show more aggressive behavior,
with shorter latency to first attack and higher frequency of attacking. No significant difference is
found between MAO A-KO mice and wild-type mice in EPM test. This information suggests that
MAO A deficiency is more likely associated with increased aggression rather than anxiety, and
this aggressive behavior is probably linked to significantly elevated levels of serotonin and
dopamine.
1.2 Generation of MAO B-KO mouse, alteration of substrates, and reduced
anxiety
The MAO B knockout (MAO B-KO) mouse is generated through the insertion of a
transcriptionally active neomycin resistance gene into exon 6 of MAO B gene. Once the
targeting construct (pMAO B-KO) has recombined with the MAO B allele, a stop codon is
generated, creating a truncated and inactive MAO B enzyme (Grimsby et al. 1997). As more
gene editing strategies are being discovered, now the MAO B-KO can also be achieved through
CRISPR/Cas 9. The guide RNA targets about 13kb upstream of the start codon to about 8kb
downstream of the stop codon of the MAO B gene. This gRNA direct the Cas9 protein to induce
a site-specific double-strand break in the genomic DNA, and the non-homologous end-joining
(NHEJ) resulted in the deletion of ~ 130 kb of MAO B gene (Obata et al. 2022).
10
No significant alteration is shown on the concentrations of serotonin, dopamine,
norepinephrine, and 5-HIAA in MAO B-KO mice. Serotonin and dopamine concentrations
increased by less than 10% in MAO B knockout (KO) mice. Other substrates, such as dopamine
metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-
methoxytyramine (3-MT), and norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol
(MHPG), all showed similar level in the brains of MAO B-KO mice compared to wild-type mice
(Grimsby et al. 1997). However, MAO B-KO mice exhibited significantly elevated levels of
phenylethylamine, with an eightfold increase in the brain and a three to tenfold increase in the
striatum compared to wild-type mice (Grimsby et al. 1997) (Table 1). This suggests that
phenethylamine is the preferred substrate for MAO B.
Since serotonin and dopamine are crucial for emotion regulation, the absence of MAO B
theoretically should not induce aggression in the mice. This is supported by experimental results
(Chen et al. 2004) (Table 2). MAO B knockout mice did not display signs of aggressive
behavior. In elevated plus maze test, under the 300-lux light condition, no significant difference
was observed between wild-type and MAO B-KO mice. However, when the light condition
changed to 10 lux, MAO B-KO mice showed more entries into the open arms, as well as a longer
stay (Bortolato et al. 2009). MAO B-KO mice and wild-type showed similar entries and time of
stay in the closed arms of the maze. The difference in behavior is due to the change in light
conditions, as higher illuminance level may exaggerate the anxiety level of mice and therefore
affect the result of EPM test. In open-field test with 10 lux of light, the MAO B-KO mice
showed slightly shorter time spent inside the chamber and significantly shorter latency to leave
11
the chamber (Bortolato et al. 2009). The result of open-field test and EPM test suggests that
MAO B-KO mice exhibit reduced anxiety levels. Interestingly, in the forced swim test (FST),
which tests the reaction of rodents under unavoidable stress such as swimming, MAO B-KO
mice showed higher mobility compared to wild-type (Grimsby et al. 1997). This represents a less
depressive-like behavior and a better response to stress with MAO B deficiency.
These findings suggest that the absence of the MAO B enzyme does not affect the regulation
of aggression, but may affect the regulation of anxiety and depression.
1.3 Generation of MAO A/B-KO mouse, alteration of substrates, aggression,
and anxiety behavior
The generation of MAO A/B-KO mice is slightly more complicated. Since MAO A and
MAO B genes are too close to each other on the chromosome with only 24kb apart, MAO A/B
double knockout (MAO A/B-KO) mice cannot be bred through the breeding of MAO A-KO
mouse with MAO B-KO mouse. It is achieved through A863T mutation in MAO A exon 8 of
MAO B-KO mice (Chen et al. 2004). The mutation of adenine at position 863 into thymine in
exon 8 of the MAO A gene replaces the amino acid 284 lysine (AAA) with a stop codon (TAA)
in the sequence (Chen et al. 2004). This A863T mutation changed the sequence of the DraI
restriction site (TTTAAA to TTTTAA), resulting in the absence of MAO A transcription and
enzymatic activity on the bases of MAO B-KO mice (Scott et al. 2008).
In the MAO A/B-KO mice, increased levels of serotonin (850%), dopamine (170%),
norepinephrine (220%), and phenylethylamine (1570%) were detected. The level of 5-HIAA
decreased with a percentage of about 20,000% compared to wild-type mice, which is an even
12
lower level compared to only MAO A-KO mice (Chen et al. 2004; Thöny et al. 2024) (Table 1).
Comparing the alteration of neurotransmitters in three types of MAO-KO mice, result showed
that MAO A and MAO B have different substrate specificities. Serotonin and norepinephrine are
more preferred by MAO A, while phenylethylamine is more preferred by MAO B. Dopamine
and tyramine are common substrates of both proteins.
Table 1. The relative levels of neurotransmitters and their substrates in the brain of MAO knockout mice compared to wild-type
Alteration of neurotransmitter levels in MAO knockout mice compared to wild-type
MAO A-KO MAO B-KO MAO A/B-KO
5-HT 200% 107% 850%
NE 130% No change 220%
DA 110% 109% 170%
5-HIAA (5-HT metabolite) 50% no change 0.5%
DOPAC (DA metabolites) 28.5% 103% significantly decrease
HVA (DA metabolites) Unknown 97% Unknown
3-MT (DA metabolites) Unknown No change Unknown
MHPG (NE metabolite) Unknown No change Unknown
PEA No change 829% 1570%
Increasing chemicals are marked in red and decreasing chemicals are marked in blue. Substrates with no significant alterations
remain unhighlighted. Data for MAO A-KO is based on Chen et al. 2004; Cases et al. 1995; Bortolato et al. 2008. Data for MAO
B-KO is based on Grimsby et al. 1997. Data for MAO A/B-KO mice is based on Chen et al. 2004.
Different from the increased fighting behavior in MAO A-KO mice and reduced anxiety in
MAO B-KO mice, MAO A/B double KO mice showed more anxiety-like behavior (Scott et al.
2008). In Chen’s study (Chen et al. 2004), compared to the C57-BL/6J/129Sv strain wild-type
mice, MAO A/B-KO mice exhibited significantly reduced exploration in unfamiliar open areas
(Table 2). Their locomotor activity, as evidenced by the distance traveled in the arena area per
minute, was markedly lower (MAO A/B-KO mice: 43.51 ± 11.53 cm min–1, WT mice: 93.45 ±
13.67 cm min–1), along with decreased time spent in the central arena. In the elevated plus-maze
test, MAO A/B-KO mice demonstrated fewer entries into the maze, preferring the closed arms
over the open arms, in contrast to wild-type mice which exhibited more entries with a similar
preference for both arms. In the resident-intruder test, MAO A/B-KO resident mice displayed a
13
shorter latency to attack the intruder (MAO A/B-KO mice: 15.5 ± 6.0 s, WT mice: 533.3 ± 39.5
s), accompanied by reduced social interaction and an increased frequency of rapid chase and
escape behaviors. Interestingly, despite these differences, MAO A/B-KO mice did not exhibit
typical aggressive behaviors such as lateral threat and piloerection at frequencies differing from
those observed in wild-type mice.
Table 2. The behavioral test results of MAO KO mice compared to wild-type on aggression, anxiety, and depression
Behavioral tests on MAO knockout mice compared to wild-type
Tests MAO A-KO MAO B-KO MAO A/B-KO
aggression
Cricket killing test shorter latency to
attack Untested Untested
Resident-intruder test
Aggression toward
intruder, shorter
latency to attack, and
less social interaction
No aggression facing
intruder
Aggression toward
intruder, shorter
latency to attack, and
less social interaction
anxiety
Open area
Fewer exploration in
open area
Slightly shorter stay
in chamber
Significantly reduced
exploration in open
area
Elevated-plus maze No difference
More entries into the
open arm and longer
stay
Fewer entries into the
maze, and closed arms
preferred
depression Forced swim test Untested Higher mobility Untested
Behaviors elevated are marked in red and behaviors reduced are marked in blue. No significant differences remain unhighlighted.
Results for MAO A-KO are based on Vishnivetskaya et al. 2007; Chen et al. 2004. Results for MAO B-KO are based on Grimsby
et al. 1997; Chen et al. 2004; Bortolato et al. 2009. Results for MAO A/B-KO mice are based on Scott et al. 2008; Chen et al.
2004.
In conclusion, MAO A/B-KO mice show higher serotonin, dopamine, norepinephrine, and
PEA levels. These mice exhibit reduced exploration and locomotor activity in unfamiliar open
areas, as well as a preference for the closed arms in the EPM test. In the resident-intruder test,
they demonstrate a shorter latency to attack intruders and increased rapid chase and escape
behaviors, though without typical aggressive displays like lateral threat and piloerection. This
information confirms the relationship between MAO A deficiency and aggression, as well as
pointing out that extremely high serotonin and dopamine levels may contribute to not only
aggressive behavior but also anxiety.
14
Chapter 2. MAO A-KO mouse and MAO A/B-KO mouse show
symptoms of autism spectrum disorder
Autism spectrum disorder (ASD) is a neurological disease with symptoms of impaired
communication and social features, repetitive behavior, and inflexible behaviors (APA 2000).
This disease may be due to the abnormal connectivity between cerebral and cerebellar cortices.
Previous research found that an increased serotonin level appeared in about 30% to 50% of ASD
patients (Anderson et al. 1987). Therefore, the role serotonin plays in ASD is studied through the
MAO A-KO mouse model.
2.1 MAO A-KO mouse exhibits ASD features in behavioral test and
neuropathology analysis
In the study by Bortolato (Bortolato et al. 2013), multiple behavior tests were done. Social
interaction and social investigation were tested to see the social features and communication of
the mice. The T-maze test, hole-board test, and marble burying test were done to test whether the
mice have repetitive behavior. The water maze, sticky tape test, and beam-walking test were
designed to test whether the mice have cognitive and behavior inflexibility.
In the social interaction test, both the test mouse and a foreign male mouse with similar ages
and weights to the test mouse were placed into an unfamiliar cage. The social features of the test
mouse were recorded. MAO A-KO mice exhibited fewer social interactions with the foreign
mouse, with greatly decreased frequency and duration of anogenital sniffing, less following, and
less grooming behavior compared to the wild-type. The digging, locomotion, or sniffing of the
abdominal area did not show much decrease in MAO A-KO group. In the social investigation
15
test, a foreign male mouse was placed inside a cylindrical wire cup in the center of the cage. The
test mouse was then placed in the corner of the cage so that it can investigate around. The
reaction of the test mouse to the foreign mouse was recorded. The number and duration of
investigative approaches to the foreign mouse both decreased in MAO A-KO mice (Table 3).
Repetitive behavior is one of the most important features of ASD. It is determined by the test
results of the T-maze test, hole-board test, and marble burying test. T-maze test apparatus is
shaped like a letter “T”, with the bottom end as the starting point and the top two arms as the
goal arms. The mouse is put in the starting compartment, and a successful test trial is done if the
mouse chooses either of the arms within 120 seconds. A total of eight trials were done and the
arm alternation was recorded. If the mouse shows a tendency to explore a new arm, which means
higher alternation in this test, this mouse is defined as with good cognitive function of learning
and memory. In MAO A-KO mice, a low tendency of alternation was observed.
Hole-board test is another test that focuses on repetitive behaviors. The apparatus had 16
holes in it, and the total number of times that the mouse pokes its head into the hole was
recorded. The exploration of the same hole is considered repetitive behavior. In MAO A-KO
mice, the number of head pokes greatly decreased, with the majority of the pokes toward the
same hole. The low alternation confirms the repetitiveness.
The marble burying test places a mouse in the cage covered with sawdust and 24 glass
marbles. The duration of digging and the number of marbles buried are recorded. MAO A-KO
group displayed higher marble-burying and digging activity than wild-type mice, which suggests
higher repetitive behavior and anxiety level.
16
The inflexible cognition is then tested through a water maze test. The Morris water maze is a
large circular tub divided into quadrants and filled with nontransparent water. A small platform is
placed in the middle of the quadrant. The mouse first goes through visual training and spatial
training, in which the mouse is placed in the pool and must swim to find the hidden platform.
After several days of training, the animal is able to learn the location of the platform using spatial
memory. Later in the reversal learning, which tests the mouse’s ability to adapt to change, the
platform is placed in the adjacent quadrant and the time that the mouse spent in the quadrant with
the target platform is recorded. MAO A-KO mice show similar learning time to wild-type in
special training. However, when getting to the reversal learning phase, MAO A-KO mice did not
show significantly reduced time to reach the platform as wild-type mice did. The time MAO AKO mice spent in the target quadrant is shorter as well, indicating a failure of learning.
Sensorimotor is tested through sticky tape test and beam-walking test. Sticky tape test
records the time that the mouse takes to remove the previously set piece of tape from the
forepaw, while beam-walking test records the number of foot-slips the mouse made when
walking on a metal rod. Results of these tests show MAO A-KO mice exhibited increased tape
removal time in the sticky tape test and increased foot-slips in beam-walking test. The crawling
posture and gait deficits when walking down the beam also supported the lack of sensorimotor in
MAO A-KO mice.
In the neuropathology analysis, MAO A-KO mice showed neurological changes consistent
with ASD features, such as reduced thickness of the corpus callosum, increased dendritic
arborization of pyramidal neurons in the prefrontal cortex, and disrupted microarchitecture of the
17
cerebellum. The thickness of corpus callosum was greatly decreased in MAO A-KO, indicating a
deficiency in the connection between the frontal cortex and prefrontal cortex. The dendritic
arborization of pyramidal neurons in the prefrontal cortex increased 33% in number of branches
and 38% in total length.
2.2 MAO A/B-KO mouse exhibits ASD features in behavioral test and
neuropathology analysis
Similar to MAO A-KO mice, MAO A/B-KO mice showed ASD symptoms in the behavioral
tests (Bortolato et al. 2013) (Table 3).
Table 3. The behavioral test results of MAO KO mice compared to wild-type on autism spectrum disorder
Behavioral test on MAO knockout mice compared to wild-type
Features Tests MAO A-KO MAO B-KO MAO A/B-KO
Social feature and
communication
Social interaction Fewer social
interaction Untested Fewer social
interaction
Social investigation Fewer investigative
approach Untested Fewer investigative
approach
Repetitive behavior
T-maze test Lower tendency of
alteration Untested Lower tendency of
alteration
Hole-board test Fewer head-pokes
and low alteration
Shorter latency to
first head poke
Fewer head-pokes,
fewer exploration, and
low alteration
Marble burying test
Higher marbleburying and digging
activity
Reduced marbleburying activity
Higher marbleburying and digging
activity than MAO AKO
Cognitive and
behavior inflexibility
Water maze
Longer time spent to
reach the platform
and shorter time spent
in the target quadrant
Untested
Reduced locomotor
activity and shorter
time spent in the
target quadrant than
MAO A-KO
Sticky tape test Increased tape
removal time Untested Increased tape
removal time
Beam-walking test Increased foot-slips Untested Increased foot-slips
Neuropathology analysis Reduced thickness of
the corpus callosum Untested
Reduced thickness of
the corpus callosum
compared to MAO AKO
Behaviors elevated are marked in red and behaviors reduced are marked in blue. Results for MAO A-KO are based on Bortolato
et al. 2013. Results for MAO B-KO are based on Bortolato et al. 2009. Results for MAO A/B-KO mice are based on Bortolato et
al. 2013.
In the social interaction test, MAO A/B-KO mice showed greatly decreased frequency and
18
duration of anogenital sniffing, less following, and less grooming behavior compared to wildtype, while in social investigation test, the number and duration of investigative approach to the
foreign mouse both decreased in MAO A/B-KO mice.
The T-maze test and hole-board test were done to test whether the mice have repetitive
behavior. Same as MAO A-KO, a lower number of alternations was observed in MAO A/B-KO
mice. The number of entries to the arm of the first choice was even higher in MAO A/B-KO.
This suggests a highly repetitive behavior. In hole-board test, the exploration and hole-poking
decreased, with a decreased rate of alternation. In marble-burying test, even though both MAO
A-KO and MAO A/B-KO displayed higher burying and digging activity than wild-type, MAO
A/B KO showed significantly higher marble-burying activity than MAO A KO mouse.
The water maze, sticky tape test, and beam-walking test were designed to test whether the
mice have cognitive and behavior inflexibility. In the reversal learning phase of the water maze
test, the MAO A/B-KO mice showed significantly reduced locomotor activity but longer distance
traveled compared to both MAO A-KO and wild-type. The time MAO A/B-KO mice spent in the
target quadrant is even shorter compared to MAO A-KO. The behavioral tracking graph showed
that MAO A/B-KO had no preference for the quadrant with the platform, which means no spatial
memory of past learning and cognition inflexibility. The increased tape removal time in sticky
tape test and increased foot-slips in beam-walking test supported this result. The crawling
posture and gait deficits when walking down the beam also supported the lack of sensorimotor in
MAO A/B-KO mice.
In the neuropathology analysis of MAO A/B-KO mice, the reduction in the thickness of
19
corpus callosum was greater compared to MAO A-KO. The dendritic arborization of pyramidal
neurons in the prefrontal cortex increased 26% in number of branches and 23% in total length.
MAO A/B KO mice had a significant reduction in Purkinje cells and an increase in molecular
layer thickness, but not granule layer thickness compared with wild-type controls. These
mutations indicated a disrupted connectivity of the cerebellar cortex. The severity of repetitive
behaviors and neuropathological alterations is generally greater in MAO A/B knockout mice
compared to those with only MAO A knocked out.
The study suggested that the neurochemical imbalances caused by MAO A deficiency, either
alone or in combination with MAO B deficiency, lead to abnormalities that resemble those seen
in ASDs. The severity of these symptoms is greater in MAO A/B-KO mice, highlighting the
combined impact of both enzyme deficiencies. The study underscores the critical role of MAO A
in regulating behaviors and brain structures associated with ASD.
Chapter 3. MAO A-KO mouse shows suppressed growth of prostate
cancer and melanoma
3.1 MAO A-KO cells reduce ROS production and reverse EMT in prostate
cancer
In the previous sections, the mechanism of monoamine deamination is presented, in which
reactive oxygen species are created as a byproduct. ROS leads to lipid peroxidation that breaks
up the cell membrane and induces apoptosis, as well as oxidation of nucleobase and amino acids
20
that cause DNA damage and protein malfunction (Shields et al. 2021). These cellular damages
contribute to various diseases, including tumorigenesis.
In Wu’s study, the effect of ROS and MAO A gene on prostate cancer (PCa) is well discussed
(Wu et al. 2014). MAO A influences the hydroxylation process of prolyl hydroxylase domains 1-
4 (PHD1-4) by regulating ROS levels. PHD1-4 enzymes hydroxylate two proline residues on the
transcription factor hypoxia-inducible factor 1-alpha (HIF-1α), which signals it for recognition
and ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, leading to its
degradation by the proteasome.
Increased ROS levels inhibit the activity of PHD enzymes, thereby stabilizing HIF-1α under
normoxic conditions. HIF-1α is a key mediator of the cellular response to hypoxia and also
regulates the VEGFA and its pathway. The activation of VEGF’s co-receptor, neuropilin-1,
promotes the AKT/FOXO1/TWIST1 signaling pathway, which is activated in high-grade
prostate cancer specimens. This pathway leads to the epithelial-to-mesenchymal transition
(EMT), a process where cells lose their polarity and adhesion properties and gain migratory and
invasive capabilities, increasing the potential for metastasis (Wu et al. 2014) (Figure 3).
Furthermore, EMT is associated with the development of resistance to chemotherapy and
targeted therapies in cancer cells, complicating treatment efforts. By understanding the impact of
ROS and MAO A on HIF-1α and related pathways, new therapeutic strategies might be
developed to better manage prostate cancer, especially in overcoming treatment resistance and
preventing metastasis.
21
Figure 3. Reactive oxygen species (ROS) triggering Epithelial-to-mesenchymal transition pathway. Adapted from Wu et al. 2014.
The mechanisms described above were validated through in vitro studies using human
prostate cancer cell lines. The PC-3 cell line, characterized by low MAO A expression, and the
LNCaP cell line, characterized by high MAO A expression, were employed in this research. The
study first examined the role of MAO A in stabilizing HIF-1α. Under normal oxygen conditions,
HIF-1α levels were found to be higher in MAO A-overexpressing PC-3 cells compared to the
control group. When these cells were cultured under hypoxic conditions, the MAO Aoverexpressing cells exhibited a faster and greater degree of HIF-1α stabilization. Additionally,
there was an upregulation of EMT-promoting genes such as SNAIL2, TWIST1, VEGFA, and
GLUT1. Conversely, in MAO A-KO LNCaP cells, the expression of these genes were
downregulated, and the stability of HIF-1α decreased, demonstrating that higher MAO A
22
expression leads to increased HIF-1α stability.
The study also focuses on the relationship between MAO A and EMT. When MAO A was
overexpressed in PC-3 cells, a change in morphology to a dispersed type occurs. Western blot
analysis revealed a significant decrease in E-cadherin (an epithelial marker) and an increase in
vimentin (a mesenchymal marker), N-cadherin, and TWIST1. This overexpression also led to
increased cancer cell invasiveness. Conversely, knocking out MAO A in LNCaP cells using
MAO A-targeting shRNA increased E-cadherin expression and decreased vimentin and Ncadherin levels, reducing cancer cell invasiveness. The observed decrease in E-cadherin and
increase in vimentin markers indicated that high MAO A expression in cancer cells promotes
characteristics of the epithelial to mesenchymal transition, suggesting MAO A's involvement in
EMT regulation.
These conclusions were further supported by in vivo xenograft mouse model studies using
C57BL/6 mice. LNCaP, C4-2, ARCaPM, and MPC3 prostate cancer cells with either MAO A-KO
or wild-type, were implanted into the mice. The tumor growth was determined in each group. In
the MAO A-KO group, tumors were smaller in size and grew more slowly compared to the wildtype mice. Remarkably, ARCaPM and MPC3 cells in the MAO A-KO group exhibited no tumor
growth, whereas the wild-type group showed normal significant tumor growth. These results
suggested that MAO A plays an important role in tumor growth.
Furthermore, the MAO A-KO xenograft tumors showed a decrease in the EMT process,
evidenced by increased E-cadherin staining and decreased vimentin staining in IHC analysis.
There was also a decrease in HIF-1α and VEGFA staining, indicating that the hypoxic conditions
23
supporting tumor growth were suppressed in the MAO A-KO mice.
In summary, the study demonstrates that MAO A plays a significant role in stabilizing HIF1α and promoting EMT in prostate cancer cells, thereby enhancing tumor growth and
invasiveness.
3.2 MAO A-KO mouse show inhibited TAM polarization in melanoma
Later on, more studies on the effect of MAO-KO on cancer were conducted. Wang's study
proposed that MAO A deficient mice exhibit reduced polarization of tumor-associated
macrophages (TAMs), consequently leading to enhanced antitumor activity (Wang et al. 2021).
TAMs are specialized macrophages derived from bone marrow-derived monocytes (BMDMs).
Depending on the local microenvironment and the presence of pro-inflammatory or antiinflammatory factors such as chemokines and cytokines, monocytes undergo a process known as
macrophage polarization, resulting in differentiation into either an immunostimulatory or an
immunosuppressive phenotype. Under the influence of anti-inflammatory cytokines like IL-4
and IL-13, TAMs assume an immunosuppressive phenotype. These TAMs play a pivotal role in
shaping the immunosuppressive tumor microenvironment (TME). It is widely believed that
TAMs promote tumorigenesis and malignancy while also inhibiting T-cell activity, thereby
dampening the efficacy of various cancer therapies, including immune checkpoint blockade
therapy (Wang et al. 2021).
The polarization of TAM, however, is not an irreversible process. TAM has a special
characteristic called plasticity, which means under the effect of certain chemicals or reagents, the
immunosuppressive TAM may be subject to repolarization into the immunostimulatory
24
phenotype. One hypothesis posits that ROS may serve as stimuli for triggering the polarization
of TAMs towards an immunosuppressive state, linking this phenomenon to the activity of the
MAO enzyme. All of these macromolecular damages contribute to the initiation and
development of cancer. In the case of tumor-associated macrophage polarization, ROS exhibit as
the stimulus that triggers macrophages to polarize toward the immunosuppressive phenotype,
therefore, the MAO A-KO mouse model is used in the study of whether inhibition of MAO A
could be applied as a cancer immunotherapy targeting repolarization of TAM.
In this study, experiments were conducted on C57BL/6J mice which serve as wild-type, and
MAO A-KO mice. These mice were implanted with B16-OVA melanoma tumors to investigate
the impact of MAO A deficiency on tumor-associated macrophage (TAM) polarization and tumor
growth. qPCR analysis of isolated monocytes and TAMs from tumor-bearing mice revealed a
significant reduction in MAO A gene expression within TAMs compared to monocytes,
suggesting a role for MAO A in TAM regulation.
Furthermore, tumor growth in MAO A-KO mice was markedly suppressed compared to
wild-type mice, despite similar TAM levels. However, TAMs from MAO A-KO mice exhibited
lower expression of the immunosuppressive marker CD206 and higher levels of
immunostimulatory molecules such as CD69, CD86, and I-Ab. Additional analysis demonstrated
reduced expression of immunosuppressive genes (Mrc1, Chi3l3, Arg1) and increased expression
of pro-inflammatory cytokine genes (Il6, Tnfα, Ccl2) in TAMs from MAO A-KO mice.
Moreover, CD8+ T cells infiltrating the tumors of MAO A-KO mice showed enhanced
activation, as indicated by increased Granzyme B production.
25
To test specifically on immune cells instead of whole-body MAO A knockout, a cell transfer
was done in which bone-marrow cells from both MAO A-WT and MAO A-KO were harvested
and transferred into CD45.1 wild-type mice, followed by the B16-OVA tumor implantation. And
the results were consistent with the whole-body MAO A-KO mice. MAO A deficiency in
immune cells showed downregulation of immunosuppressive markers and upregulation of
immunostimulatory markers, resulting in reversed TAM polarization, suppressed tumor growth,
and enhanced tumor-infiltrating CD8+ T-cell activation, highlighting the direct regulatory role of
MAO A in immune cell antitumor activity.
The same result appeared when another macrophage transfer was done in which bonemarrow cells from both MAO A-WT and MAO A-KO were harvested and co-cultured with
BMDMs, and then the mixture was transferred into CD45.1 wild-type mice, followed by the
B16-OVA tumor implantation. This was to test specifically on TAM polarization, and the results
were consistent with the whole-body MAO A-KO mice.
The study revealed that MAO A plays a significant role in regulating tumor-associated
macrophage (TAM) polarization and tumor growth. MAO A deficiency in both whole-body and
immune cell-specific models led to suppressed tumor growth, decreased expression of
immunosuppressive markers, increased expression of immunostimulatory markers, and enhanced
activation of CD8+ T cells. These findings suggest that targeting MAO A could be a promising
therapeutic strategy to modulate the tumor microenvironment and boost antitumor immunity.
26
Chapter 4. Discussion
4.1 The role of MAO in neurotransmitter metabolism, aggression, and anxiety
As MAO being an enzyme that involves in the metabolism of neurotransmitters such as
serotonin, norepinephrine, dopamine, and phenylethylamine, MAO-KO mouse model had been
applied in various research, including Kevin Chen’s study on anxiety (Chen et al. 2004), Olivier
Cases’s study on aggression (Cases et al. 1995), Bortolato’s research on ASD (Bortolato et al.
2009), Jason Boyang Wu’s study on prostate cancer (Wu et al. 2014), and Yu-Chen Wang’s study
on melanoma (Wang et al. 2021), as discussed in this paper.
MAO A-KO mice showed an increased concentration of serotonin (200%), dopamine
(110%), and norepinephrine (130%) compared to wild-type, while MAO B-KO mice showed a
slightly increased concentration of serotonin (107%), dopamine (109%), and a greatly increased
phenylethylamine (829%). In MAO A/B-KO mice, levels of all monoamine neurotransmitters
increased drastically, with 850% of serotonin, 220% norepinephrine, 170% dopamine, and
1570% phenylethylamine. Even though phenylethylamine is not an MAO A preferred substrate
and serotonin is not an MAO B preferred substrate, the increase of concentration in MAO A/BKO mice that were way higher than single KO suggests that MAO A may still play a role in PEA
metabolism, so does MAO B in serotonin degradation.
The research on anxiety and aggression elucidated how neurobiological and social behaviors
are intertwined. Excessive aggression and anxiety were linked to impaired emotion regulation
and disrupted neuroendocrine function, leading to problematic social behaviors. Comparing the
experiment results from Chen, both MAO A-KO mice and MAO A/B-KO mice showed fewer
27
exploration attempts and shorter stays in open areas. This may indicate a higher level of anxiety
when MAO A enzyme is not present. MAO A single knockout mice showed no significantly
different behavior from wild-type mice in the EPM test, while MAO B single knockout mice
showed longer stay in the open arm. However, MAO A/B double KO mice showed fewer entries
into the maze, and a preference to closed arm than open arm. In RI test, MAO A-KO mice
showed no difference from wild-type when facing immobile intruders or juvenile intruders.
However, when facing intruders with similar ages and body sizes, no matter how aggressive the
intruder is, MAO A-KO mice would show more aggressive behavior, with shorter latency to first
attack and higher frequency of attacking. Similar to MAO A-KO mice, MAO A/B-KO mice
showed shorter latency to attack as well. However, the MAO A/B-KO mice did not perform
typical aggressive behaviors when facing intruders. This information confirmed the relationship
between MAO A deficiency and aggression, as well as pointing out that extremely high serotonin
and dopamine levels may contribute to both aggressive behavior and anxiety.
In previous studies, serotonin is considered to be a substrate that regulates impulse
aggression. Low serotonin levels have been related to aggressive behaviors. In the MAO A-KO
and MAO A/B-KO mouse models, however, the high serotonin level is associated with
aggression. One possible explanation is that the serotonin concentration in MAO A-KO mice is
too high that the serotonin receptors become desensitized, leading to the downregulation of
serotonin transmission. Both too high or too low serotonin levels would cause aggression,
suggesting the possibility of a U-shaped relationship between aggression and serotonin levels.
28
4.2 The role of MAO A in regulating behaviors and brain structures
associated with autism spectrum disorder
The high aggression and anxiety levels in MAO A-KO mice and MAO A/B-KO mice are
then further extended to the hypothesis of ASD. The study by Bortolato investigated the
behavioral and neuropathological characteristics of MAO A-KO and MAO A/B-KO mice to
understand the role of MAO A deficiency in ASD. Both MAO A-KO and MAO A/B-KO mice
exhibited ASD-like symptoms, including impaired social interaction, repetitive behavior, and
cognitive inflexibility. These behavioral abnormalities were confirmed through various tests such
as social interaction and investigation tests, T-maze test, hole-board test, water maze test, sticky
tape test, and beam-walking test.
In MAO A-KO mice, decreased social interactions, high repetitive behaviors, and cognitive
inflexibility were observed, alongside neurological changes like reduced corpus callosum
thickness, increased dendritic arborization in the prefrontal cortex, and disrupted cerebellar
microarchitecture. Similarly, MAO A/B-KO mice showed even more pronounced ASD-like
behaviors and neuropathological alterations, including a significant reduction in corpus callosum
thickness, increased dendritic branching, and disrupted cerebellar cortex connectivity. In
contrast, MAO B-KO mice did not exhibit ASD features, as evidenced by a reduced amount of
marble-burying activity on both numbers and durations and shorter latency to the first head dip
in hole-board tests (Bortolato 2009). This suggested that the deficiency in MAO B, which
primarily affects phenylethylamine levels, does not significantly impact the neurotransmitter
systems involved in ASD-related behaviors. These findings indicated that MAO A deficiency,
29
whether alone or in combination with MAO B deficiency, leads to neurochemical imbalances
and structural brain changes that contribute to ASD-like symptoms. The severity of these
symptoms was greater in MAO A/B-KO mice, highlighting the combined impact of both enzyme
deficiencies. The study underscores the critical role of MAO A in regulating behaviors and brain
structures associated with ASD.
4.3 MAO A and potential therapeutic implications in neuroblastoma
In the study by Jason Wu, the role of MAO A in prostate cancer progression was examined,
particularly through the stabilization of HIF-1α and the promotion of epithelial to mesenchymal
transition (EMT). The research demonstrated that MAO A influences the hydroxylation process
of prolyl hydroxylase domains (PHD1-4) by regulating reactive oxygen species (ROS) levels,
which in turn stabilizes HIF-1α under normoxic conditions. Additionally, HIF-1α regulates the
VEGFA pathway, promoting the AKT/FOXO1/TWIST1 signaling pathway, which is activated in
high-grade prostate cancer specimens and leads to EMT, enhancing metastasis and treatment
resistance.
The in vitro studies using human prostate cancer cell lines, PC-3 and LNCaP, highlighted that
MAO A overexpression results in higher HIF-1α levels and upregulation of EMT-promoting
genes. Conversely, MAO A knockout (KO) in LNCaP cells decreased the expression of these
genes and reduced HIF-1α stability, indicating the significant role of MAO A in EMT regulation
and cancer cell invasiveness. This was further supported by in vivo experiments using C57BL/6
mice implanted with prostate cancer cells. MAO A-KO mice exhibited smaller and slowergrowing tumors, and in some cases, no tumor growth at all, along with a reversal of the EMT
30
process and suppression of hypoxic conditions supportive of tumor growth. These findings
suggest that MAO A is a critical factor in prostate cancer progression through its role in HIF-1α
stabilization and EMT promotion. Targeting MAO A could be a promising therapeutic strategy to
inhibit EMT and tumor progression.
In the study done by Yuchen Wang, the critical role of MAO A in regulating tumor-associated
macrophage (TAM) polarization and tumor growth was discussed. Utilizing both whole-body
and immune cell-specific MAO A knockout (KO) models, the research demonstrates that MAO
A deficiency leads to a marked suppression of tumor growth in mice implanted with B16-OVA
melanoma tumors. The experiments revealed that TAMs from MAO A-KO mice exhibited
reduced expression of immunosuppressive markers (such as CD206) and increased levels of
immunostimulatory molecules (such as CD69, CD86, and I-Ab). Additionally, there was a
downregulation of immunosuppressive genes and an upregulation of pro-inflammatory cytokine
genes within TAMs from MAO A-KO mice. The findings were further corroborated through cell
transfer experiments, where bone marrow cells from both MAO A-WT and MAO A-KO mice
were harvested and transferred into wild-type mice. Consistent results showed that MAO A
deficiency in immune cells suppressed tumor growth, altered TAM polarization, and enhanced
the activation of tumor-infiltrating CD8+ T cells. This study suggests that targeting MAO A
could be a promising therapeutic strategy to modulate the tumor microenvironment and enhance
antitumor immunity.
In the previously mentioned study, the impact of MAO A on epithelial-to-mesenchymal
transition (EMT) was tested on prostate cancer, and how MAO A affects tumor-associated
31
macrophage (TAM) polarization was examined in melanoma. However, these mechanisms are
also hypothesized to be effective in other cancers, such as neuroblastoma. Neuroblastoma is a
cancer that develops in neuroblasts, immature nerve cells that typically mature into neurons or
glial cells (Sada & Tumbar 2013). When neuroblasts mutate and fail to mature properly, they
undergo uncontrolled division, leading to tumor formation. Neuroblastoma is the most common
extracranial solid tumor in children, taking up about 8-10% of pediatric cancers (Schleiermacher
2014). It originates in the adrenal medulla or sympathetic ganglia and often metastasizes to bone
marrow, regional lymph nodes, liver, or subcutaneous tissues in approximately 50% of cases
(Schleiermacher 2014).
Neuroblastoma comprises two main cell types: mesenchymal and adrenergic phenotypes (van
Groningen et al.2017). Mesenchymal neuroblastoma cells are known to exhibit resistance to
immunotherapy. Within the tumor microenvironment, under the influence of pro-tumorigenic
cytokines such as TGF-beta1, IL-6, IL-8, and various growth factors, adrenergic neuroblastoma
cells can transition into mesenchymal cells (Louault 2022). This transition is facilitated by TAMs
and mesenchymal stromal cells/cancer-associated fibroblasts, which contribute to cytokine
secretion within the tumor microenvironment.
It is hypothesized that monoamine oxidase inhibitors (MAOIs) might reduce the
immunosuppressive polarization of TAMs, thereby inhibiting the transition from adrenergic to
mesenchymal cells. This could potentially serve as a therapeutic strategy for treating
neuroblastoma by targeting the tumor microenvironment and preventing the development of
immunotherapy-resistant mesenchymal neuroblastoma cells.
32
In conclusion, research into the role of MAO A and MAO B isoenzymes in neurobiology has
provided significant insights into how these genes influence aggression, anxiety, ASD, and
cancers. MAO A/B-KO mouse models are applied in the studies, revealing the absence of these
enzymes can lead to marked changes in neuroendocrine function, affecting the regulation of
neurotransmitter levels. Understanding the in vivo function of MAO enzyme and the molecular
mechanisms is crucial for developing therapeutic strategies to address emotional disturbances
and social dysfunction in related human disorders, as well as developing better treatment for
those affected by chemotherapy resistance in cancer treatment.
33
Bibliography
Anderson, G. M., Freedman, D. X., Cohen, D. J., Volkmar, F. R., Hoder, E. L., McPhedran, P.,
Minderaa, R. B., Hansen, C. R., & Young, J. G. (1987). Whole blood serotonin in autistic
and normal subjects. Journal of Child Psychology and Psychiatry, 28(6), 885–900.
https://doi.org/10.1111/j.1469-7610.1987.tb00677.x
APA. Diagnostic and Statistical Manual of Mental Disorders DSM-IV-TR. Washington, DC:
American Psychiatric Association; 2000.
Bach, A. W., Lan, N. C., Johnson, D. L., Abell, C. W., Bembenek, M. E., Kwan, S. W.,
Seeburg, P. H., & Shih, J. C. (1988). cDNA cloning of human liver monoamine oxidase A
and B: molecular basis of differences in enzymatic properties. Proceedings of the National
Academy of Sciences of the United States of America, 85(13), 4934–4938.
https://doi.org/10.1073/pnas.85.13.4934
Bortolato, M., Chen, K., & Shih, J. C. (2008). Monoamine oxidase inactivation: From
pathophysiology to therapeutics. Advanced Drug Delivery Reviews, 60(13–14), 1527–
1533. https://doi.org/10.1016/j.addr.2008.06.002
Bortolato, M., Godar, S. C., Alzghoul, L., Zhang, J., Darling, R. D., Simpson, K. L., Bini, V.,
Chen, K., Wellman, C. L., Lin, R. C., & Shih, J. C. (2013). Monoamine oxidase A and
A/B knockout mice display autistic-like features. The international journal of
neuropsychopharmacology, 16(4), 869–888. https://doi.org/10.1017/S1461145712000715
Bortolato, M., Godar, S., Davarian, S. et al. Behavioral Disinhibition and Reduced Anxiety-like
Behaviors in Monoamine Oxidase B-Deficient Mice. Neuropsychopharmacol 34, 2746–
2757 (2009). https://doi.org/10.1038/npp.2009.118
Brunner, H. G., Nelen, M., Breakefield, X. O., Ropers, H. H., & Van Oost, B. A. (1993).
Abnormal behavior associated with a point mutation in the structural gene for monoamine
oxidase a. Science, 262(5133), 578–580. https://doi.org/10.1126/science.8211186
Cases, O., Seif, I., Grimsby, J., Gaspar, P., Chen, K., Pournin, S., Müller, U., Aguet, M.,
Babinet, C., Shih, J. C., & De Maeyer, E. (1995). Aggressive behavior and altered
amounts of brain serotonin and norepinephrine in mice lacking MAO A. Science,
268(5218), 1763–1766. https://doi.org/10.1126/science.7792602
Chen, K., Holschneider, D. P., Wu, W., Rebrin, I., & Shih, J. C. (2004). A spontaneous point
mutation produces monoamine oxidase a/b knock-out mice with greatly elevated
monoamines and anxiety-like behavior. Journal of Biological Chemistry, 279(38), 39645–
39652. https://doi.org/10.1074/jbc.M405550200
34
Drechsel, D. A., & Patel, M. (2008). Role of reactive oxygen species in the neurotoxicity of
environmental agents implicated in Parkinson’s disease. Free Radical Biology and
Medicine, 44(11), 1873–1886. https://doi.org/10.1016/j.freeradbiomed.2008.02.008
Grimsby, J., Chen, K., Wang, L. J., Lan, N. C., & Shih, J. C. (1991). Human monoamine
oxidase A and B genes exhibit identical exon-intron organization. Proceedings of the
National Academy of Sciences of the United States of America, 88(9), 3637–3641.
https://doi.org/10.1073/pnas.88.9.3637
Grimsby, J., Toth, M., Chen, K., Kumazawa, T., Klaidman, L., Adams, J. D., Karoum, F., Gal,
J., & Shih, J. C. (1997). Increased stress response and β–phenylethylamine in MAO B–
deficient mice. Nature Genetics, 17(2), 206–210. https://doi.org/10.1038/ng1097-206
Ito A, Kuwahara T, Inadome S, Sagara Y. Molecular cloning of a cDNA for rat liver
monoamine oxidase B. Biochem Biophys Res Commun. 1988;157:970–76.
Kuwahara T, Takamoto S, Ito A. Primary structure of rat monoamine oxidase A deduced from
cDNA and its expression in rat tissues. Agric Biol Chem. 1990;(54):253–57.
Lan, N. C., Heinzmann, C., Gal, A., Klisak, I., Orth, U., Lai, E., Grimsby, J., Sparkes, R. S.,
Mohandas, T., & Shih, J. C. (1989). Human monoamine oxidase A and B genes map to
xp11.23 and are deleted in a patient with norrie disease. Genomics, 4(4), 552–559.
https://doi.org/10.1016/0888-7543(89)90279-6
Louault, K., Porras, T., Lee, M.-H., Muthugounder, S., Kennedy, R., Sarte, L., Fernandez, G.
E., Pawel, B., Shimada, H., Asgharzadeh, S., & Declerck, Y. A. (2022). Abstract 3123:
Cancer-associated fibroblasts and tumor-associated macrophages cooperate to promote
TGF-β1-dependent NFkB activation and IL6 production and immune escape. Cancer
Research, 82(12_Supplement), 3123–3123. https://doi.org/10.1158/1538-7445.AM2022-
3123
MAO A gene: Medlineplus genetics. (2017). Accessed 2024 May 18, Available from:
https://medlineplus.gov/genetics/gene/MAO A/
Maoa monoamine oxidase a [homo sapiens (Human)]—Gene—NCBI. Bethesda (MD):
National Library of Medicine (US), National Center for Biotechnology Information; 2004
– [cited 2024 July 16]. Available from: https://www.ncbi.nlm.nih.gov/gene/4128
Maob monoamine oxidase b [homo sapiens (Human)]—Gene—NCBI. Bethesda (MD):
National Library of Medicine (US), National Center for Biotechnology Information; 2004
– [cited 2024 July 16]. Available from: https://www.ncbi.nlm.nih.gov/gene/4129
35
Neumann. (2010). Aggression and anxiety: Social context and neurobiological links. Frontiers
in Behavioral Neuroscience. https://doi.org/10.3389/fnbeh.2010.00012
Obata, Y., Kubota-Sakashita, M., Kasahara, T., Mizuno, M., Nemoto, T., & Kato, T. (2022).
Phenethylamine is a substrate of monoamine oxidase B in the paraventricular thalamic
nucleus. Scientific Reports, 12(1), 17. https://doi.org/10.1038/s41598-021-03885-6
Sada, A., & Tumbar, T. (2013). New insights into mechanisms of stem cell daughter fate
determination in regenerative tissues. International Review of Cell and Molecular Biology
(volume 300, page 1–50). Elsevier. https://doi.org/10.1016/B978-0-12-405210-9.00001-1
Schleiermacher, G., Janoueix‐Lerosey, I., & Delattre, O. (2014). Recent insights into the
biology of neuroblastoma. International Journal of Cancer, 135(10), 2249–2261.
https://doi.org/10.1002/ijc.29077
Scott, A. L., Bortolato, M., Chen, K., & Shih, J. C. (2008). Novel monoamine oxidase A knock
out mice with human-like spontaneous mutation. NeuroReport, 19(7), 739–743.
https://doi.org/10.1097/WNR.0b013e3282fd6e88
Segura‐Aguilar, J., Paris, I., Muñoz, P., Ferrari, E., Zecca, L., & Zucca, F. A. (2014). Protective
and toxic roles of dopamine in Parkinson’s disease. Journal of Neurochemistry, 129(6),
898–915. https://doi.org/10.1111/jnc.12686
Seo, D., Patrick, C. J., & Kennealy, P. J. (2008). Role of serotonin and dopamine system
interactions in the neurobiology of impulsive aggression and its comorbidity with other
clinical disorders. Aggression and Violent Behavior, 13(5), 383–395.
https://doi.org/10.1016/j.avb.2008.06.003
Shields, H. J., Traa, A., & Van Raamsdonk, J. M. (2021). Beneficial and detrimental effects of
reactive oxygen species on lifespan: A comprehensive review of comparative and
experimental studies. Frontiers in Cell and Developmental Biology, 9, 628157.
https://doi.org/10.3389/fcell.2021.628157
Shih, J. C. (2018). Monoamine oxidase isoenzymes: Genes, functions and targets for behavior
and cancer therapy. Journal of Neural Transmission, 125(11), 1553–1566.
https://doi.org/10.1007/s00702-018-1927-8
Soubrié, P. (1986). Reconciling the role of central serotonin neurons in human and animal
behavior. Behavioral and Brain Sciences, 9(2), 319–335.
doi:10.1017/S0140525X00022871
36
Thöny, B., Ng, J., Kurian, M. A., Mills, P., & Martinez, A. (2024). Mouse models for inherited
monoamine neurotransmitter disorders. Journal of Inherited Metabolic Disease, 47(3),
533–550. https://doi.org/10.1002/jimd.12710
Van Groningen, T., Koster, J., Valentijn, L. J., Zwijnenburg, D. A., Akogul, N., Hasselt, N. E.,
Broekmans, M., Haneveld, F., Nowakowska, N. E., Bras, J., Van Noesel, C. J. M.,
Jongejan, A., Van Kampen, A. H., Koster, L., Baas, F., Van Dijk-Kerkhoven, L., HuizerSmit, M., Lecca, M. C., Chan, A., … Versteeg, R. (2017). Neuroblastoma is composed of
two super-enhancer-associated differentiation states. Nature Genetics, 49(8), 1261–1266.
https://doi.org/10.1038/ng.3899
Vishnivetskaya, G. B., Skrinskaya, J. A., Seif, I., & Popova, N. K. (2007). Effect of MAO A
deficiency on different kinds of aggression and social investigation in mice. Aggressive
Behavior, 33(1), 1–6. https://doi.org/10.1002/ab.20161
Wang, Y.-C., Wang, X., Yu, J., Ma, F., Li, Z., Zhou, Y., Zeng, S., Ma, X., Li, Y.-R., Neal, A.,
Huang, J., To, A., Clarke, N., Memarzadeh, S., Pellegrini, M., & Yang, L. (2021).
Targeting monoamine oxidase A-regulated tumor-associated macrophage polarization for
cancer immunotherapy. Nature Communications, 12(1), 3530.
https://doi.org/10.1038/s41467-021-23164-2
Wu, J. B., Shao, C., Li, X., Li, Q., Hu, P., Shi, C., Li, Y., Chen, Y.-T., Yin, F., Liao, C.-P.,
Stiles, B. L., Zhau, H. E., Shih, J. C., & Chung, L. W. K. (2014). Monoamine oxidase A
mediates prostate tumorigenesis and cancer metastasis. Journal of Clinical Investigation,
124(7), 2891–2908. https://doi.org/10.1172/JCI70982
Abstract (if available)
Abstract
Monoamine oxidase (MAO) is an enzyme involved in the metabolism of neurotransmitters such as serotonin (5-HIAA), norepinephrine (NE), dopamine (DA), and phenylethylamine (PEA). In this paper, we discuss research using MAO knockout (KO) mice that provide insights into various conditions. Studies by Olivier Cases (Cases et al. 1995) showed that MAO A knockout (KO) mouse models exhibit significantly elevated levels of serotonin, dopamine, and norepinephrine, leading to increased aggressive behavior. In contrast, MAO B KO mice, with significantly elevated phenylethylamine levels, did not display aggressive behavior but exhibit less anxiety and depressive-like behaviors in studies by Joseph Grimsby et al (Grimsby et al. 1991). Studies by Kevin Chen (Chen et al. 2004) revealed that MAO A/B-KO mice show heightened anxiety-like behavior. Study by Marco Bortolato et al. (Bortolato et al. 2008) showed that MAO A-KO and MAO A/B-KO mice exhibit pronounced features of autism spectrum disorder (ASD). Jason Boyang Wu's study on prostate cancer (Wu et al. 2014) demonstrated that MAO A promotes cancer progression through HIF-1α stabilization and epithelial-to-mesenchymal transition (EMT), suggesting MAO A as a therapeutic target. Yu-Chen Wang's research on melanoma (Wang et al. 2021) highlighted MAO A's role in regulating tumor-associated macrophage (TAMs) polarization, indicating that targeting MAO A could modulate the tumor microenvironment and improve cancer treatment outcomes. In this paper, we discuss the multifaceted role of MAO enzyme in neurobiological and oncological processes, providing us with clues of possible treatment strategies and research directions that can be applied to other diseases.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Monoamine oxidase and cancer
PDF
Monoamine oxidase inhibitors regulate tumorigenesis and mitochondrial function in a prostate cancer mouse model
PDF
MAO a deficient mice exhibit an altered immune system in the brain and prostate
PDF
Co-expression of monoamine oxidase A and prostate cancer stem cell markers in Pten knockout mice
PDF
NMI: a near infrared conjugated MAO-A inhibitor as a novel targeted therapy for colorectal and other cancers
PDF
Monoamine oxidase deficiency and emotional reactivity: neurochemical and developmental studies
PDF
Potential therapeutic effect of monoamine oxidase (MAO) inhibitor on human neuroblastoma
PDF
Monoamine oxidase A inhibitors and androgen receptor antagonists regulate mitochondrial function in prostate cancer cells
PDF
NMI (near-infrared dye conjugate MAO A inhibitor) outperformed FDA-approved prostate cancer drugs with a unique mechanism based on bioinformatic analysis of NCI60 screening data
PDF
Downregulation of osteopontin by PTEN in liver-specific Pten-null mouse model
PDF
Inhibition of MAO-A by Dual MAO-A/HDAC inhibitors: in silico approach for ligand binding and affinity prediction
PDF
The intersection of mitochondrial biology and cancer: insights from mitochondrial microproteins and mtDNA alterations
PDF
Development of engineered antibodies as novel anti-cancer agents
PDF
AKT exhibits isoform-specific function in hepatocyte transformation and response to cellular stress
PDF
Pharmacokinetic modeling: ciprofloxacin in the environment and metformin PBPK model
PDF
The role of endoplasmic reticulum chaperones in adipogenesis, liver cancer and mammary gland development
PDF
Role of purinergic P2X4 receptors in regulation of dopamine homeostasis in the basal ganglia and associated behaviors
PDF
Investigating the effects of targeting CD99 on T cells to enhance their antileukemia activity
PDF
Experience-dependent neuroplasticity of the dorsal striatum and prefrontal cortex in the MPTP-lesioned mouse model of Parkinson’s disease
PDF
Investigating the effect of FLT3 tyrosine kinase inhibitors and anti-FLT3 antibody-based therapy in acute myeloid leukemia
Asset Metadata
Creator
Gao, Zijin
(author)
Core Title
Monoamine oxidase (MAO) knock-out mouse models: Tools for studying the molecular basis of aggression, anxiety, autism and cancers
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Degree Conferral Date
2024-08
Publication Date
08/30/2024
Defense Date
08/30/2024
Publisher
Los Angeles, California
(original),
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
aggression,Anxiety,autism,cancer,MAO-KO mouse
Format
theses
(aat)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Shih, Jean Chen (
committee chair
), Tabancay, Angel P., Jr. (
committee member
), Zhang, Yong (
committee member
)
Creator Email
gaozijinaimee@gmail.com,zijingao@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC11399A0AB
Unique identifier
UC11399A0AB
Identifier
etd-GaoZijin-13455.pdf (filename)
Legacy Identifier
etd-GaoZijin-13455
Document Type
Thesis
Format
theses (aat)
Rights
Gao, Zijin
Internet Media Type
application/pdf
Type
texts
Source
20240831-usctheses-batch-1205
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Repository Email
cisadmin@lib.usc.edu
Tags
aggression
autism
cancer
MAO-KO mouse