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Characterization of mouse models of peroxisome biogenesis disorders: a study of dental enamel phenotypes

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Characterization of mouse models of peroxisome biogenesis disorders: a study of dental enamel phenotypes

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i

CHARACTERIZATION OF MOUSE MODELS OF PEROXISOME BIOGENESIS
DISORDERS: A STUDY OF DENTAL ENAMEL PHENOTYPES

by

Yudhishtar Singh Bedi

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2013

Copyright 2013 Yudhishtar Singh Bedi

ii
DEDICATION
I dedicate this document to my parents, Nandini and Harinder Bedi for their constant
support.

iii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Dr. Joseph Hacia, for taking
me on, supporting me and providing me the opportunity to break new ground in the field
of peroxisome biology with this project in the limited amount of time that I have been a
part of the lab. His words of encouragement have always motivated me even when results
don’t come out as expected. His ability to manage the effort and logistics involved in
collaborating with labs, not only within the University of Southern California (USC), but
also between countries and yet still find the time to check on my work and progress in the
lab has always amazed me. Whenever I’ve been stuck at some point during my research,
his insights have really helped figure out what my next move should be and have
strengthened this study greatly.
A special thanks to my co-advisor, Dr. Michael Paine, for teaching me everything
I know about enamel maturation and assisting me with the experimental aspects of this
thesis. Within the short time that I have spent analyzing mouse heads in his lab, he
always made sure I was comfortable in the lab. His daily check-ins and rounds have
always kept the lab a very cheerful and personable place. He’s always been the go-to
person in the lab whenever I’ve come across any blocks in terms of lab facilities required
or whenever I’ve needed advice on results that I’ve been confused about. It was certainly
a pleasure to be able to work in his lab in this collaborative effort between two labs.
I’d also like to thank Dr. Vijay Kalra, for being the third member of my thesis
committee and for always coming through whenever I have needed a chat or advice for
my career goals or even a game of tennis. It’s been amazing having him as my professor

iv
these last two years at USC. Despite his distinguished position at the University he has
always found time to address any issues I have had at an absolute informal level as my
friend, rather than as my superior.
I feel extremely blessed to have had Dr. Xin Wen as my mentor in the lab. I am
grateful for her guidance and good humor. She helped me master techniques of SEM and
microCT analysis within a matter of weeks where it would probably have taken me
months on my own. Even though she has always had a lot of work for her own research,
she always found time to make sure my work was running smoothly, especially when we
had trouble getting the software for the microCT analyses to work. Conversations with
her between experiments and the lovely classical music that played from her computer
have always kept the lab a lively, cultured and beautiful place.
I must thank Anitha Krishnan and Douglas Hauser for their support in helping me
perfect my analyses with microCT at the Molecular Imaging Center and SEM at the Cell
and Tissue Imaging Core, respectively. Their guidance has been invaluable to my studies.
Nemi Doshi has been a great colleague and lab partner to have during this entire
time. More than that he has been a great friend and support, especially whenever we
needed to get away from the lab and explore new exciting places in the city. I am deeply
grateful for his friendship and am delighted that both of us had started and now
completed our projects together.
Lastly, I’d like to thank my family and friends who have been a source of constant
support and inspiration to me and without whom this thesis would not have been
possible.

v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
INTRODUCTION 1

CHAPTER 1: LITERATURE REVIEW 3
1.1 Peroxisome 3
Metabolic functions of peroxisomes 4
Peroxisome assembly 12
1.2 Peroxisome Disorders 13
Peroxisome biogenesis disorders (PBDs) 14
Single peroxisomal enzyme or transporter protein defects 15
1.3 Mouse Models for Peroxisome Disorders 18
1.4 Enamel Defects in Peroxisomal Disorders 19
1.5 Enamel Maturation and Structure 21
Hierarchical structure of enamel 22

CHAPTER 2: RESEARCH METHODOLOGY 24
2.1 Mice Obtained for Analysis 24
2.2 Microcomputed Tomography 24
2.3 Scanning Electron Microscopy 25

CHAPTER 3: RESULTS AND DISCUSSIONS 28

CHAPTER 4: CONCLUSION 35

BIBLIOGRAPHY 37

vi

LIST OF TABLES

Table 1: Chemical properties of ROS/RNS produced in peroxisomes 5

Table 2: Enzymes in mammalian peroxisomes that generate ROS/RNS 5

Table 3: Enzymes in mammalian peroxisomes that degrade ROS/RNS 5

Table 4: List of mice used for analyses 24

Table 5: Reagents used to prepare Eponate Resin 25

vii
LIST OF FIGURES

Figure 1. Schematic overview of peroxisomal enzymes that produce or
decompose reactive oxygen species (ROS) 6 6

Figure 2. Schematic overview of representation of steps involved in
biosynthesis of plasmalogens with topology of enzymes involved 8

Figure 3. Fatty acid β-oxidation between peroxisome and mitochondria 9

Figure 4.
a) Enzymatic steps of alpha-oxidation
b) Schematic illustration and topology of the enzymes involved in
the phytanic acid alpha-oxidation pathway in mammalian
peroxisomes 10

Figure 5. Different reactions carried out by peroxisomal Polyamine
oxidase (PAO) 12

Figure 6. Targeting signals used by peroxisomal proteins. 12

Figure 7. Model for the early stages in the import of peroxisomal matrix
proteins 13

Figure 8. X-linked Adrenoleukodystrophy (X-ALD) as a
metabolic/inflammatory syndrome 16

Figure 9. Role of alpha-methylacyl-CoA racemase in peroxisome 17

Figure 10. Preoperative intraoral photographs of patient with IRD 20

Figure 11. Clinical photographs of IRD patient’s occlusion and dentition
prior to treatment, showing occlusal wear 21

Figure 12. Comparison of incisors of mice with Pex1 G844D mutation on
both alleles to heterozygote 28

Figure 13. More comparisons of mutant vs. heterozygote incisors under
light microscope 29

Figure 14. MicroCT images of molars of Pex1 mutant vs. wild-type 30

Figure 15. MicroCT images of incisor and 1
st
molar of Pex1 mutant vs.
wildtype 30

viii

Figure 16. MicroCT images of molars of Pex7 mutant vs. wild-type 31

Figure 17. MicroCT images of incisor and 1
st
molar of Pex7 mutant vs.
wildtype 32

Figure 18. SEM images showing crystal microstructure of enamel
Pex1 WT vs. Pex1 mutant 33

Figure 19. SEM images showing crystal microstructure of enamel of
Pex7 WT mice vs. mutants 34

ix
ABSTRACT

This thesis describes a collaborative investigatory effort between the labs of Dr. Joseph
Hacia (USC), Dr. Michael Paine (USC), Dr. Steven Steinberg (Kennedy Krieger Institute,
Baltimore) and Dr. Nancy Braverman (McGill University, Canada) to understand the link
between human peroxisome biogenesis disorders and enamel defects in patients carrying
these diseases.
We, at the Hacia lab, received skulls or parts of skulls of Pex1 mice with gene disruptions
on one or both alleles and knock out Pex7 mice from Dr. Steinberg’s and Dr.
Braverman’s lab respectively and carried out a series of analyses to inspect the micro and
nano structures of the teeth of these mice. These investigations were carried out in Dr.
Paine’s lab and involved microcomputed tomography (µCT) and scanning electron
microscopy (SEM) analyses of overall structure of dentition and fine structure of
hydroxyapatite crystals in the prisms and interprismatic regions of enamel.
Our studies concluded that, at least for these specific Pex1 and Pex7 genetic mutations,
there is no discernible difference in the structure of the enamel of these animals.

1
INTRODUCTION
Peroxisomes disorders refer to a general class of medical conditions caused by
impaired peroxisome function(s) (Wanders and Waterham 2006). These disorders can
have a serious impact on human health, given that peroxisomes are essential for a
variety of essential functions including fatty acid (FA) beta-oxidation, plasmalogen
biosynthesis and detoxification of reactive oxygen species (ROS). Since plasmalogen
is an essential component of myelin, a common target for peroxisomal disorders is the
nervous system. Symptoms of these disorders range from neuro-developmental delay,
loss of auditory and visual senses, severe growth deficiencies and craniofacial
abnormalities to severe hypotonia, liver and kidney dysfunction, mental retardation
and frequently results in death within the first year of life (Raymond GV 2001).
Peroxisome biogenesis defects form a separate group in the family of peroxisome
disorders. These are caused due to mutations in at least 13 PEX genes that code for
peroxin proteins responsible for proper assembly of the peroxisome (Levesque S et al
2012). Thus, as the name suggests, these disorders cause the lack of an intact
peroxisome. The spectrum of disorders within this subgroup range in severity such
that children who inherit the severe type of the disease, e.g. Zellweger syndrome, die
before one year of age, whereas those who inherit the milder form of the disease, e.g.
Infantile Refsum Disease, may continue to live till adulthood but with a reduced
quality of life. While research continues to find hard cures for these diseases that may
use stem cells to correct the mutations at a genetic level (Wang XM et al 2012), a lot
can also be done to help people who are currently living with such disorders to
improve their lifestyles so they may live as close to a normal life as possible. In recent

2
years, many families with children and family members who carry these diseases
have found each other either via the Internet (thegfpd.org, rhizokids.com) or through
the clinics and have formed support groups to better understand the disease. Such
groups help each other out and even raise awareness and potential funding
opportunities. Including reports from these families, independent case reports have
been published in the literature about children with severe enamel defects amongst
other craniofacial anomalies (Tran D et al 2011, Acharya BS et al 2012). Currently
there is little literature that attempts to solve why disorders of peroxisomes cause
these enamel defects and even less mention of the appropriate management strategies
of such problems.
Within the last decade, mouse models have been generated for peroxisomal disorders
by knocking out genes of the PEX family (Wanders and Waterham 2006). These have
been shown to successfully mimic the human disorders and they present a subset of
the human clinical phenotypes. With these animal models much has been added to the
knowledge about peroxisomes as well as the diseases that are caused by their lack or
malfunction by studying mice models. With the intent of finding out why PBDs cause
enamel defects, this study attempts to investigate the enamel structural phenotypes of
mice generated with inactivated Pex1 and Pex7 genes. Future studies may also be
done in mice carrying additional PEX mutations to understand the biochemical
pathways controlled by peroxisomes leading to defects in amelogenesis and enamel
formation.

3
CHAPTER 1: LITERATURE REVIEW
1.1 Peroxisome
Along with glyoxysomes and glycosomes, peroxisomes are members of the
microbody family. This essential class of cell organelles is ubiquitous in all
eukaryotes and is characterized by the presence of a proteinacious matrix surrounded
by a single membrane. After being described by a Swedish doctoral student, J Rhodin,
in 1954, it was not until 1967 that they were identified as cell organelles by Belgian
cytologist Christian de Duve (de Duve C 1969).
Peroxisomes are involved in fatty acid beta-oxidation as well as several other
functions that are not common to peroxisomes in lower eukaryotes such as FA alpha-
oxidation, ether phospholipid biosynthesis and glyoxylate detoxification.
Wanders and Waterham (2006) describe three major breakthroughs that enhanced our
knowledge about peroxisomes within the last 40 years as:
1. Identifying Yeast mutants defective in peroxisome biogenesis.
• Characterization of these mutants led to the discovery of two distinct
peroxisomal targeting signals (PTSs – PTS1 and PTS2).
• Using this knowledge, computer based searches for proteins with the
PTS1 or PTS2 sequence could be performed and mammalian
orthologues were identified using homology probing.
2. Identifying a class of genetic diseases in man.

4
• Genetic defects in which peroxisome biogenesis or peroxisome
functions are defective have given us new insights into the metabolic
roles carried out by peroxisomes in humans.
3. Discovery of closed structure of peroxisomes.
• As opposed to the earlier belief that peroxisomes were freely
permeable, it was discovered that they were, in fact, closed structures
in vivo.
• This led to the discovery of a set of half ATP-binding cassette (ABC)
transporters that might be involved in transmembrane FA transport.
1.1.1 Metabolic functions of peroxisomes
1. Oxygen Metabolism, ROS and RNS Metabolism
A number of oxidases (Table 2) that reside in peroxisomes cause reduction of Oxygen
to Hydrogen peroxide (Schrader M et al 2004), one of the several reactive species
generated in peroxisomes (Table 1). Other enzymes (Table 3) also present in
peroxisomes, namely, catalase, glutathione peroxidase and peroxiredoxin V (PMP20)
can then decompose this hydrogen peroxide (Singh AK et al 1994, Yamashita H et al
1999). The oxidase form of Xanthine oxidoreductase is also responsible for creating
highly reactive superoxide anions. These in turn are inactivated by superoxide
dismutases. Nitric oxide (NO) species are generated by nitric oxide synthase activity
(Stolz DB et al 2002) that in turn react with superoxides to form very highly reactive
peroxynitrites (Fig 1). These peroxynitrites are reduced by peroxiredoxin V that has
peroxynitrite reductase activity (Dubuisson M et al 2004). Peroxisomes also possess

5
epoxide hydrolase activity as well as Glutathione S-transferase activity (Waechter F
et al 1983, Morel F et al 2004).
Name Formula Biological half-life
Membrane
permeability
1. Hydrogen peroxide H
2
O
2
~10
−5
s

Very low
2. Superoxide radical O
2
•
−
~10
−6
s Very low
3. Hydroxyl radical •OH ~10
−9
s Very low
4. Nitric oxide radical NO• < 1 s High
5. Peroxynitrite ONOO
–
~ 1 s Very low

Table 1: Chemical properties of ROS/RNS produced in peroxisomes (M Fransen et al 2012)
Enzyme Substrate PTS
ROS/RNS
generated
1. Acyl-CoA oxidases
a. Palmitoyl-CoA oxidase
b. Pristanoyl-CoA oxidase
c. Trihydroxycoprostanoyl-CoA
oxidase
Long chain fatty acids
2-Methyl branched-chain fatty acids
Bile acid intermediates
PTS1 H
2
O
2
2. D-Amino acid oxidase D-Proline PTS1 H
2
O
2

3. D-Aspartate oxidase D-Aspartate, N-methyl-D-aspartate PTS1 H
2
O
2

4. α-Hydroxyacid oxidase Glycolate, lactate PTS1 H
2
O
2

5. Pipecolic acid oxidase L-Pipecolic acid PTS1 H
2
O
2

6. Polyamine oxidase N-Acetyl spermine/spermidine PTS1 H
2
O
2

7. Urate oxidase Uric acid PTS1 H
2
O
2

8. Xanthine oxidase Xanthine U
H
2
O
2
, NO•,
O
2
•
−

9. Inducible nitric oxide synthase L-Arginine U NO•, O
2
•
−

PTS1 = Peroxisomal targeting signal 1; U = Unknown; H
2
O
2
= Hydrogen peroxide; NO• = Nitric oxide
radical; O
2
•
−
= Superoxide radical

Table 2: Enzymes in mammalian peroxisomes that generate ROS/RNS (Schrader and Fahimi
2004, M Fransen et al 2012)

Enzyme Substrate Enzyme also present in
1. Catalase H
2
O
2
Cytoplasm and nucleus (some cells)

2. Glutathione peroxidase H
2
O
2
All cell compartments
3. Mn SOD O
2
•
− Mitochondria
4. Cu, Zn SOD O
2
•
− Cytoplasm
5. Epoxide hydrolase Epoxides Cytoplasm and ER
6. Peroxiredoxin V
H
2
O
2
, ONOO
–
,
ROOH
Cytoplasm and nucleus
SOD = Superoxide dismutase; H
2
O
2
= Hydrogen peroxide; NO• = Nitric oxide radical; O
2
•
−
=
Superoxide radical; ONOO
–
= Peroxynitrite; ROOH = Hydroperoxide; ER = Endoplasmic Reticulum

Table 3: Enzymes in mammalian peroxisomes that degrade ROS/RNS (Schrader and Fahimi
2004, M Fransen et al 2012)

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Figure 1. Schematic overview of peroxisomal enzymes that produce or decompose reactive
oxygen species (ROS). Hydrogen peroxide is produced by a number of peroxisomal oxidases (for
example by acyl-CoA oxidase which is involved in the beta-oxidation of fatty acids). Hydrogen
peroxide is decomposed by catalase and glutathione-peroxidase (GPx) or converted to hydroxyl
radicals. Hydroxyl radicals can damage the peroxisomal membrane by lipid peroxidation of
unsaturated fatty acids. Hydroperoxides formed in this process can be decomposed by catalase
and glutathione-peroxidase. Superoxide anions (O
2
·
–
) generated by peroxisomal oxidases are
scavenged by manganese superoxide dismutase (MnSOD) and by copper-zinc superoxide
dismutase (CuZnSOD). Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine (L-
Arg) to nitric oxide (NO·). NO· can react with O
2
·
–
radicals to form peroxynitrite (ONOO
–
), a
powerful oxidant. (Schrader and Fahimi 2004)
2. Ether-Phospholipid Biosynthesis
Ether phospholipids exist as plasmanyl-phospholipids and plasmenyl-phospholipids
(plasmalogens) with either ethanolamine or choline as head groups. Almost one-fifth
of phospholipid mass in humans is made up of plasmalogens with high levels
occurring in the brain, heart, kidney, spleen, skeleton, muscles and testis (Wanders JA

7
2006). These plasmalogens are also present in abundance in myelin. Thus,
deficiencies in plasmalogens affect myelination of nerve cells, thereby affecting the
entire nervous system. Alkyldihydroxyacetone phosphate synthase (ADHAPS) carries
out the first committed step of ether-linked glycerolipid synthesis by forming Alkyl-
DHAP from Acyl-DHAP (Fig 2), replacing the ester-linked FA with an ether-linked
fatty alcohol. While ADHAPS is an established peroxisomal enzyme, the enzyme that
creates its substrates is also peroxisomal. DHAP acyltransferase (DHAPAT)
generates Acyl-DHAP from DHAP and an acyl-CoA ester (Thai TP et al 1997).
DHAPAT-deficient human and CHO cell lines cannot synthesize plasmalogens (Liu
D et al 2005) making DHAPAT highly crucial for plasmalogen synthesis.

8

Figure 2. Schematic overview of representation of steps involved in biosynthesis of plasmalogens
with topology of enzymes involved (Image from Peroxisome database: www.peroxisomedb.org).
Enzymes are
1) Dihydroxyacetone phosphate acyltransferase (DHAPAT)
2) Alkyldihydoxyacetone phosphate synthase (ADHAPS)
3) Fatty acyl-CoA reductase (FAR)
4) Glycerol-3-phosphate dehydrogenase (G3PD)
5) Acylalkyl-dihydroxyacetone phosphate reductase (AADHAPR)
6) Peroxisomal transporter PMP34
7) Very long chain acyl-CoA synthase (VLCS)
ER: Endoplasmic Reticulum, DHA: Dihydroxyacetone

3. Peroxisomal FA Beta-Oxidation
While short-, medium- and most long-chain FAs are beta-oxidized in the
mitochondria, very long chain FAs (VLCFAs), C26:0 in particular (Cerotic acid or
Hexacosanoic acid, 26 carbon long chain saturated fatty acid i.e. with 0 double
bonds), can only be beta-oxidized by peroxisomes (Fig 3). Other substrates include
pristanic acid (derived from phytanic acid alpha-oxidation), di- and tri-

9
hydroxycholestanoic acid (DHCA and THCA) (from cholesterol in liver), long-chain
monocarboxylic acids, certain PUFAs, certain prostaglandins and leukotrienes, some
xenobiotics and vitamins E and K (Wanders and Waterham 2006).

Figure 3. Fatty acid β-oxidation between peroxisome and mitochondria (Dr. JA Illingworth,
Lectures on Metabolism, University of Leeds)
4. Peroxisomal FA Alpha-Oxidation
The presence of an alkyl group at the beta carbon of a fatty acyl-CoA blocks its beta-
oxidation. These FAs with methyl at C3 position must go through alpha-oxidation
first to its (n-1) FA. The process of alpha-oxidation involves hydroxylation of the
alpha carbon, removal of the terminal carboxyl group and linkage of CoA to the
terminal carboxyl group. This branched substrate will function in the beta-oxidation
process, ultimately yielding propionyl-CoA, acetyl CoAs and, in the case of phytanic
acid, 2-methyl propionyl CoA. Phytanic acid is an example of such a fatty acid (Fig
4a). Most studies are performed with phytanic acid, which is known to accumulate in

10
peroxisomal disorders such as Refsum disease where alpha oxidation is
compromised (Wanders 2006).

Figure 4 a) Enxymatic steps of
alpha-oxidation
(www.wikipedia.org)

Figure 4 b) Schematic illustration
and topology of the enzymes
involved in the phytanic acid
alpha-oxidation pathway in
mammalian peroxisomes
(Wanders and Waterham 2006)

11
5. Glyoxylate Metabolism
Alanine:glyoxylate aminotransferase (AGT) found in liver peroxisomes converts
glyoxylate formed in peroxisomes into glycine using alanine as an amino group donor
thus preventing buildup of toxic oxalate, an end product of glyoxylate (Hoppe B
2012). AGT deficiency causes buildup of oxalate crystals in liver and kidney causing
kidney failure and loss. Hyperoxaluria type I can also be caused by mislocation of
AGT to mitochondria (Danpure CJ et al 1993).
6. Pentose Phosphate Pathway (PPP)
The PPP involves generation of NADPH by oxidation of glucose-6-phosphate to
ribose-5-phosphate (Lehninger Principles of Biochemistry 5
th
edition, 2008). About
one-tenth of the total activity of the two PPP enzymes, glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase, occurs in the peroxisome
(Antonenkov VD 1989). It is believed that these two enzymes provide the
intraperoxisomal NADPH needed for various reactions (Wanders 2006).
7. Polyamine Oxidation
Polyamines like spermine (SPM) and spermidine (SPD) are essential for a number of
vital cellular functions such as wound healing, tissue growth and differentiation and
also in tumor growth. The peroxisomal enzyme Polyamine oxidase (PAO) is key in
the regulation of intracellular polyamine concentration (Wanders and Waterham
2006). It also acts as a determinant of cellular sensitivity to antitumor polyamine
analogs (Peroxisome database, peroxisomedb.org). N
1
-acetyl-polyamines and SPM
are substrates for PAO (Fig 5).

12

Figure 5. Different reactions carried out by peroxisomal Polyamine oxidase (PAO) (Peroxisome
database 2.0)

1.1.2 Peroxisome assembly
Peroxisomes can be generated from the ER and replicate themselves by fission
(Hoepfner D 2005). Specific Peroxisomal targeting signals (PTS), PTS1 (a conserved
C-terminal tripeptide sequence) or PTS2 (a nonapeptide sequence located near the N-
terminus or at internal locations in proteins), direct proteins for peroxisomal import
after they are translated in the cytoplasm (Fig 6, S Subramani et al 2000).

Figure 6. Targeting signals used by peroxisomal proteins. The PTSs are located in the boxes
along with the consensus sequences, where applicable, and conserved variants are shown below
these sequences (S Subramani et al 2000)

At least 32 peroxisomal proteins, called peroxins, are required for proper peroxisome
assembly. PEX genes encode these peroxins. The PTS signals on matrix proteins are

13
recognized by soluble PTS receptors in the cytosol; Pex5p for PTS1 proteins
(McCollum D et al 1993) and Pex7p for PTS2 proteins (Marzioch M et al 1994).
Docking proteins, Pex13p and Pex14p, each of which form distinct complexes with
Pex5p and Pex7p allow for docking of these receptor-cargo complexes at the
peroxisomal membrane (S Subramani et al 2000).

Figure 7. Model for the early stages in the import of peroxisomal matrix proteins (S Subramani
et al 2000)

1.2 Peroxisome Disorders
Peroxisomal disorders can be broadly categorized under two groups (Wanders 2006)

14
1. Peroxisome Biogenesis Disorders (PBDs) or disorders of peroxisome assembly
2. Single peroxisomal enzyme or transport protein deficiencies
In the first category, abnormalities in multiple peroxisomal enzymes exist due to
which the peroxisome fails to form. In the second category, peroxisomal structure is
intact, however, single protein abnormalities exist.
1.2.1 Peroxisome biogenesis disorders
The PBDs are further subdivided into Zellweger spectrum disorders (ZSDs) and
rhizomelic chondrodysplasia punctata (RCDP).
1. Zellweger Spectrum Disorders
Also called as cerebrohepatorenal syndrome, Zellweger syndrome is characterized by
complete absence of peroxisomes. This occurs due to mutations in at least 12 different
PEX genes (Weller S et al 2005, Shimozawa N et al 2004). Other disorders included
within this spectrum are Neonatal Adrenoleukodystrophy (NALD) and Infantile
Refsum Disease (IRD). The order of severity of the disorder decreases from ZS to
NALD to IRD. While all three present common symptoms of liver disease, variable
neuro-developmental delay, retinopathy and perceptive deafness, ZS patients are
usually hypotonic and die before they are a year old. NALD patients present neonatal
onset hypotonia and seizures, and they have progressive white matter disease leading
to death in late infancy (Raymond GV 2001). IRD patients, on the other hand, may
survive beyond infancy and perhaps even reach adulthood.

15
These disorders are characterized by accumulation of substrates that would
normally be handled by peroxisomes, for instance: VLCFAs, pristanic acid, phytanic
acid, Dihydroxycholestanoic acid (DHCA), Trihydroxycholestanoic acid (THCA) and
pipecolic acid. Another characteristic biochemical outcome in these patients would be
low levels of peroxisome metabolism end products, e.g., plasmalogens, cholic and
chenodeoxycholic acid, and docosahexaenoic acid.
2. Rhizomelic Chondrodysplasia Punctata (RCDP), Type 1
Mutations in the PEX7 gene that encodes for the PTS2 receptor cause RCDP1
(Braverman et al 1997). This disease is characterized by a significant reduction in
plasmalogen levels due to impaired plasmalogen synthesis, dysfunctional oxidation of
phytanic acid with increasing levels in plasma and the presence of 3-oxoacyl-CoA
thiolase precursor. Patients usually display disproportionate shortening of proximal
parts of extremities, dysmorphic facial appearance, ocular aberrations, growth
deficiency and mental retardation (Huffnagel I et al 2013). It is inherited as an
autosomal-recessive disorder with limited treatment.
1.2.2 Single peroxisomal enzyme or transporter protein defects
1. X-linked Adrenoleukodystrophy (X-ALD) and Adrenomyeloneuropathy (AMN)
This disorder is characterized by mutations in ABCD1 gene that encodes for the ABC
transporter of FAs (Raymond GV 2001). Patients exhibit accumulation of VLCFAs
(Fig 8). Childhood cerebral form of ALD (CCALD) is the most common form of this
disorder, affecting boys who develop normally till 4-8 years of age followed by rapid
deterioration and death within two years (Wanders 2006).

16

Figure 8. X-ALD as a metabolic/inflammatory syndrome (Galea E et al 2012)
2. Acyl-CoA Oxidase (ACOX1) Deficiency
Mutations in ACOX1 gene leading to hypotonia, early onset seizures, hearing loss,
retinopathy and neurological abnormalities with elevated VLCFAs (Raymond GV
2001).
3. D-Bifunctional Enzyme Deficiency
HSD17B4 gene mutation leads to DBP deficiency causing Zellweger-like symptoms.
Peroxisomal beta-oxidation is impaired which results in accumulation of VLCFAs,
pristanic acid,DHCA and THCA in tissues and plasma (Baes M et al 2000)
4. α-Methylacyl-CoA Racemase (AMACR) deficiency

17
This disorder leads to accumulation of pristanic acid, DHCA and THCA (Fig 9)
with symptoms of slow, progressive vision loss, neurological deterioration and
hepatopathy (Ferdinandusse S et al 2012).

Figure 9. Role of alpha-methylacyl-CoA racemase in peroxisome. Schematic representation of the
steps involved in the oxidation of (3R)- and (3S)-phytanic acid as derived from dietary sources
and (25R)-THCA produced from cholesterol in the liver. After the activation of (3R)- and (3S)-
phytanic acid to their corresponding CoA esters, they both become substrates for the
peroxisomal alpha-oxidation system, which produces (2R)- and (2S)-pristanoyl-CoA. As
branched chain acyl-CoA oxidase, the first enzyme of the beta-oxidation system can only handle
the (S)-stereoisomer, (2R)-pristanoyl-CoA needs to be converted by AMACR into its (2S)-
stereoisomer. The bile acid intermediates, DHCA and THCA, are exclusively produced as (25R)-
stereoisomers. To be beta-oxidized, the CoA esters of the (25R)-stereoisomer also need to be
converted to the (25S)-stereoisomer by AMACR (Ferdinandusse S et al 2012).
5. RCDP2 and RCDP3
Mutations in GNPAT and AGPS gene respectively, affecting peroxisomal enzymes
DHAPAT and ADHAPS, respectively. Patients display low plasmalogen levels as
both enzymes play a vital role in ether-phospholipid biosynthesis. Prognosis includes
rhizomelia, severe mental and physical growth retardation and early death (Wanders
2006).

18
6. Adult Refsum Disease (ARD)
Phytanic acid accumulates to toxic levels due to mutation in gene encoding
phytanoyl-CoA hydroxylase (Wanders RJA et al 2001).
7. Hyperoxaluria Type 1
It is caused by mutations in the gene encoding alanine:glyoxylate aminotransferase.
Accumulating glyoxylate leads to high levels of oxalate, which precipitates in kidneys
as calcium oxalate leading to loss of kidney function (Hoppe B 2012).
7. Acatalasaemia
The Catalase-encoding gene is mutated with an increased tendency to develop oral
gangrene (Wanders and Waterham 2006)
1.3 Mouse Models of Peroxisome Disorders
The number of patients with specific peroxisomal disorders is relatively small (these
are rare genetic disorders where the real number of incidence is unknown) and the
majority of these defects usually lead to early death (Wanders 2006). Biochemical
analyses for these disorders are mostly done on cells of these patients, especially
primary skin fibroblasts. Much valuable insight has been gained by these analyses,
however, such investigations have not helped ascertain how or why a particular
peroxisomal defect would lead to the specific pathophysiology associated with it. This
provided the rationale to generate mouse models to better understand these diseases.
Most mice modeled on specific human peroxisomal disorders display the biochemical
and phenotypical defects noted in the human cases with identical mutations (Baes M

19
1999). Some mice that have been generated for deficiencies of enzymes that do not
have a correlated identified human disease may help gain new insights into their
human counterparts.
A few shortcomings (Wanders and Waterham 2006) working with mice as models
are:
1. Certain enzymes are required for very specific conditions and deficiencies of
them may not present any outwardly obvious phenotype until those conditions
exist, e.g., Scp2(-/-) mice display biochemical and phenotypic defects only
after being fed with the phytanic acid precursor phytol
2. The much shorter life span of mice as compared to humans may also prove a
hindrance in studies, as the timeframe required for development of
phenotypical abnormalities may be more than the limits of the life span of the
animal.
1.4 Enamel Defects in Peroxisomal Disorders
Within the last few years at least two case reports have been published describing
enamel defects in children with IRD (Tran D et al 2011, Acharya et al 2012). Prior to
these studies, there has not been adequate documentation relating to dental
manifestations and appropriate management of such patients.
The first report published in 2011 (Tran D et al 2011) explains the case of a 15-year
old girl with IRD who has pathological dental development, including the delayed
exfoliation of primary teeth, microdontia and hypoplastic enamel (Fig 10). Other
craniofacial anomalies listed for this affected individual were: high forehead,

20
hypoplastic supraorbital ridges, epicanthal folds, midface hypoplasia and large
anterior fontanelle. An interesting observation was the apparent smoothness and lesser
discoloration of the primary first molars and primary cuspids of the patient as
compared to her other affected teeth.

Figure 10. Preoperative
intraoral photographs taken
in the operating room. (a)
Frontal view displaying
discolored and widely spaced
teeth, with uneven edges from
attrition. Smooth-hard
enamel presents like
amelogenesis imperfecta. (b)
The maxillary arch —
hypoplastic, poorly formed
molars and cavitations on the
occlusal surfaces of teeth B, I
and the partial eruption of
teeth nos. 3 and 14. (c) The
mandibular arch also
displays partial eruption of
teeth nos. 19 and 30. (d) A
lateral view from the right.
(e) A lateral view from the
left. (Tran D et al 2011)

Another similar case was reported in 2012 (Acharya et al 2012) of a 12-year old
female patient with several unerupted permanent teeth and yellow colored enamel
(Fig 11).

21

Figure 11. Clinical
photographs of patient’s
occlusion and dentition prior
to treatment, showing
occlusal wear (Acharya et al
2012)

These reports also make mention of a cohort study of 31 IRD patients (Poll-The et al
2004) where 33% children had delayed eruption, enamel hypoplasia and
malpositioned teeth, as well as of an independent report by the mother of the 15-year
old patient stating that most IRD patients in her support group had similar symptoms
of enamel defects.
1.5 Enamel Maturation and Structure
Enamel maturation implies “the crystal growth (or expansion) that causes physical
hardening of the enamel prior to its appearance in the oral cavity on the crowns of
erupting teeth” (Smith et al 1998). This developmental step that starts, in humans, in
the third trimester of pregnancy, causes gradual hardening of soft, newly formed
enamel into one of the most durable mineralized tissues produced biologically. Full
mineralization of the primary dentition is achieved by six months after birth but may

22
take up to 4-5 years to complete on the crowns of some teeth in permanent
dentition (Moradian-Oldak J 2012).
The specialized epithelial cells that secrete enamel are called ameloblasts. These cells
dictate and demarcate the location and overall thickness of the extracellular layer as a
protein rich matrix filled with thin, ribbon-like crystals of carbonated hydroxyapatite
(Smith et al 1998). After organizing the initial crystals as rod and interrod territories,
the rod crystals lengthen with the appositional movement of the ameloblasts away
from dentin surface. A series of morphological changes at enamel surface follows
wherein tight junctions with membrane infoldings appear as ruffle-ended surfaces at
the apical ends of cells alternating with smooth ended surfaces for short intervals.
This is accompanied by changes in the net pH of the covered enamel from mildly
acidic at the ruffle-ended surfaces to near physiologic at the smooth-ended surfaces.
Matrix proteins are degraded by action of proteinases allowing the enamel crystals to
grow into these spaces occupied formerly by the protein matrix.
1.5.1 Hierarchical structure of enamel (Moradian-Oldak J 2012)
1. Microscale level prisms of enamel 50-70 nm in width and 20-25 nm in
thickness are believed to be the product of a single ameloblast and have
roughly the same diameter as these cells.
2. Interwoven arrangements of prisms (rods) with interprismatic (inter-rod)
enamel. The rods and inter-rods have different orientations with “rod-sheath”
organic material in the narrow spaces between them.

23
3. At the level of hundreds of micrometers, the interwoven prisms with
interprismatic enamel can be observed under an optical microscope as
alternating layers known as Hunter-Schreger bands.
4. A 2.5 mm thick layer of enamel covers the entire tooth.

24
CHAPTER 2: RESEARCH METHODOLOGY
2.1 Mice obtained for Analyses
Mouse
ID
Part of skull
received
Mutation
Age
sacrificed
Lab obtained
from
0006 Jaw Pex1-G844D (+/+) 28 days Steinberg*
0007 Jaw Pex1-G844D (+/+) 28 days Steinberg
0008 Jaw Pex1-G844D (-/+) 28 days Steinberg
0012 Hemimandible Pex7 (-/-) 4 months Braverman
§

0013 Hemimandible Pex7 (+/+) 1 month Braverman
0015 Hemimandible Pex7 (-/-) 4 months Braverman
0019 Hemimandible Pex7 (-/+) 4 months Braverman
0023 Head Pex1-G844D (+/+) 139 days Steinberg
0024 Head Pex1-G844D (-/+) 139 days Steinberg

Table 4: List of mice used for analyses.
*Kennedy Krieger Institute, Johns Hopkins University
§
McGill University, Canada

2.2 Microcomputed Tomography
Mice skulls were processed for microCT data collection at the Molecular Imaging
Center, Department of Radiology at Keck School of Medicine, University of Southern
California.
Data collected were then analyzed using the Amira Visage software version 5.2.0.

25
2.3 Scanning Electron Microscopy
PROTOCOL
I. Eponate Resin (EPON) embedding of teeth
i) Dissected teeth were bench dried overnight
ii) EPON was prepared in 50mL conical tube using reagents
Reagents used
Estimated volumes
(50 mL EPON)
Resin* 25.7 mL
DDSA 9.3 mL
NMA 16.5 mL
DMP-30 0.8 mL
DDSA = Dodecenyl Succinic Anhydride, NMA = Nadic Methyl
Anhydride, DMP-30 = 2,4,6-Tris[(dimethyllamino)methyl]-phenol
*TED PELLA Inc. Eponate 12 Resin was used for these
experiments.

Table 5: Reagents used for preparing Eponate Resin

Preparation was mixed for 45 min by gently rotating tube on a tube rotator at
slow speed to prevent air bubbles.
iii) 60% EPON was prepared by adding EPON to 10 mL glass bottle (used for
paraffin infiltration) and diluting with propylene oxide (CH
3
CHO). Mixed by
rotating gently for 10 min.
iv) Individual teeth were added to 10 mL glass bottles filled with EPON and
mixed gently for one hour allowing for paraffin infiltration. After one hour

26
EPON was discarded safely in EPON waste glass jar and mixing was
repeated with fresh EPON.
v) After discarding EPON from previous step, teeth were added to 60% EPON
and again mixed for one hour. If bubbles were generated, sample was allowed
to sit under fume hood with cap of bottle open to get rid of air bubbles.
vi) 100% EPON was added from flask to rubber embedding mold holder to the
top. Tooth was added to mold.
vii) Mold holder was placed in oven at 60-65°C and allowed to cure for 36-48h.
viii) Blocks were stored at RT.
II. Grinding of sample
i) Blocks were ground using coarse sandpaper till surface of interest was reached
ii) Further polishing was done using micro-cut silicon carbide grinding paper
p4000 (2.5 microns)
iii) Brushing was done frequently to remove debris during polishing finally
followed by cleaning with running water and brush.
iv) Ground blocks were air-dried.
III. Acid etching
Blocks with exposed tooth surface were acid etched in 1% HNO
3
for 45s,
rinsed with water and then, air-dried.

27
IV. Mounting the sample
i) Resin blocks were stuck to the surface of SEM carbon mounts
ii) Colloidal silver was applied to small area of sample connecting it to the mount
to prevent charge buildup under the e
-
beam of microscope
iii) Gold particles were deposited as a thin layer over the sample using the
vacuum chamber gold depositor.
The scanning electron microscope housed at the Cell and Tissue Imaging Core,
Doheny Eye Institute, University of Southern California was used to capture all SEM
images.

28
CHAPTER 3: RESULTS AND DISCUSSION
The first step before performing any experimental analyses on the mutant mice was to
do a physical observation under an optical microscope of the mice teeth, specifically,
the incisors. Observations were made for PEX1 G844D mutant and heterozygote mice
with the first batch of skulls that were received from the Steinberg lab. A difference
was initially observed with the yellow color of the incisors in that the heterozygotes
had the normal coloration, whereas the mutants had none (Fig 12). This was attributed
to the Iron deposit that normally occurs on mice incisors during development (Wen
and Paine 2013) and it was perceived that the mutants, perhaps, were flawed in this
process.

Figure 12. Comparison of incisors of mice with PEX1 G844D mutation on both alleles to
heterozygote

29
Further analyses were done on other mice skulls, also received from the Steinberg
lab. In these, the difference in color was not observed (Fig 13). The initial difference
in color was attributed to the way in which these skulls were disinfected and fixed.
This data alone may suggest greater variation in enamel color could be observed in
the Pex1G844D mutants, but a greater number of animals and qualitative data would
be needed to confirm any difference.

Figure 13. More comparisons of mutant vs. heterozygote incisors under light microscope
After a simple physical observation of the mice teeth, microCT analyses were done on
these mice using the in-house imaging facilities at the Molecular Imaging Center at
USC to check the thickness of enamel and whether there were any irregularities in the
structure of the teeth of the Pex1 and Pex7 mutants.
Although the Pex1 mutants appeared to be slightly smaller than their heterozygote
counterparts, there were no significant differences in the structure of the teeth or the
comparative thickness of the enamel (Figs 14 and 15).

30
a) Pex1 heterozygote (wild-type) b) Pex1 G844D homozygote (mutant)

Figure 14. MicroCT analyses images of molars of a) Pex1 wild-type mouse vs. b) Pex1 mutant
mouse in axial plane (above) and sagittal plane (below)

a) Pex1 heterozygote (wild-type) b) Pex1 G844D homozygote (mutant)

Figure 15. MicroCT analyses images of incisor and 1
st
molar of a) Pex1 wild-type mouse vs. b)
Pex1 mutant mouse in coronal plane (above) and sagittal plane (below, incisor only)

31
Similarly, in the Pex7 mutants, we were unable to observe any discernible
differences between null mutants and WT (Figs 16 and 17). All features of the teeth in
terms of shape and large-scale structure seemed normal. To observe ultrastructure of
enamel to check whether there were any anomalies, Scanning electron microscopy
was used to have a closer look at these teeth.

a) Pex7 heterozygote (wild-type) b) Pex7 null homozygote (mutant)

Figure 16. MicroCT analyses images of molars of a) Pex7 wild-type mouse vs. b) Pex7 mutant
mouse in axial plane (above) and sagittal plane (below)

32
a) Pex7 heterozygote (wild-type) b) Pex7 null homozygote (mutant)

Figure 17. MicroCT analyses images of incisor and 1
st
molar of a) Pex7 wild-type mouse vs. b)
Pex7 mutant mouse in coronal plane (above) and sagittal plane (below, incisor only)

The SEM housed at the Cell and Tissue Imaging Core at USC was used to obtain the
following data. The enamel ultrastructure of Pex1 mutants seemed normal and no
apparent anomalies were observed (Fig 18).

33
Pex1 heterozygote (WT) Pex1 G844D homozygote (mutant)
3000X

10000X

Figure 18. SEM images showing crystal microstructure of enamel of a) Pex1 wild-type mouse vs.
b) Pex1 mutant mouse at 3000X (above) and 10000X (below) magnifications

Analysis of Pex7 mutants also yielded no obvious distinction between null mutants,
heterozygotes and WT mice teeth (Fig 19).

34
3000X 10000X
a)
Pex7
(+/+)
WT

b)
Pex7
(+/-)
WT

c)
Pex7 (-
/-)
mutan
t

Figure 19. SEM images showing crystal microstructure of enamel of a) Pex7 wild-type mouse,
b) Pex7 heterozygote mouse with single allele mutation and c) Pex7 mutant mouse at 3000X
(left) and 10000X (right) magnifications

The figures show ordered enamel structure with seemingly normal rod and interrod
structures at 3000X magnification. At higher magnification of 10000X, individual
prisms of enamel crystals can be observed with normal structure across Pex1 and
Pex7 mutants.

35
CHAPTER 4: CONCLUSION
After obtaining data about enamel structure and thickness from microCT and SEM
techniques, the results seem to exhibit no obvious difference between Pex1 and Pex7
mutants and their respective WT or heterozygote counterparts. Aside from the
observation that the Pex1 mutants were slightly smaller than heterozygotes, the
enamel seems normal, even at the level of prismatic structure of rods and interrods.
The Pex1 mutant mice at 28 days are noticeably smaller than their wild type
counterparts, thus the smaller teeth are proportional to their body size.
Since these are observations made in mutants of only two individual PEX genes out of
the 13 confirmed genes (Levesque S et al 2012, Ebberink MS et al 2012) that are
known to be responsible for Zellweger Spectrum of disorders, one future direction to
go from here would be to investigate other Pex gene mutant animals for their enamel
and craniofacial structure.
Secondly, the developmental processes that contribute to enamel maturation in
humans take a significantly large amount of time up to one year after birth and may
sometimes even 4-5 years on the dentition of some permanent teeth. By comparison,
mice have a much shorter lifespan and an even shorter time requirement for enamel
maturation and tooth development. It is possible therefore, that not enough time is
allowed for the phenotypical abnormalities of enamel maturation to develop in mice
as they do in humans. Also, by the time gradual accumulation of VLCFAs, phytanic
acid etc. reach toxic levels, enamel maturation may already have completed.

36
Lastly, as observed by Dr. Michael Paine, the permanent dentition in humans
affected by PEX gene mutations is more severely impacted than the primary dentition
(personal conversation). Mice, however, have a single dentition and this difference
may partially explain why mutant mice have a normal, or near normal, enamel
structure. In affected humans it was also noted that the primary dentition eruption was
uneventful, while a significant delay in eruption was observed for the secondary
dentition. As rats also do not have a secondary permanent dentition (Moss-Salentijn L
1975) and are monophyodont (having only one set of teeth during their life), a similar
outcome may be expected were this study to be done in rats as test subjects. One way
to tackle this would be to perform studies on enamel in animals that are diphyodont
i.e. have two sets of teeth, one deciduous followed by a permanent dentition. The
house shrew or Suncus Murinus is one such species currently being used in the lab to
study the diphyodont replacement of deciduous dentition by replacements and
additional permanent teeth (Yamanaka A et al 2010).

37
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Abstract (if available)

Abstract This thesis describes a collaborative investigatory effort between the labs of Dr. Joseph Hacia (USC), Dr. Michael Paine (USC), Dr. Steven Steinberg (Kennedy Krieger Institute, Baltimore) and Dr. Nancy Braverman (McGill University, Canada) to understand the link between human peroxisome biogenesis disorders and enamel defects in patients carrying these diseases. ❧ We, at the Hacia lab, received skulls or parts of skulls of Pex1 mice with gene disruptions on one or both alleles and knock out Pex7 mice from Dr. Steinberg’s and Dr. Braverman’s lab respectively and carried out a series of analyses to inspect the micro and nano structures of the teeth of these mice. These investigations were carried out in Dr. Paine’s lab and involved microcomputed tomography (μCT) and scanning electron microscopy (SEM) analyses of overall structure of dentition and fine structure of hydroxyapatite crystals in the prisms and interprismatic regions of enamel. ❧ Our studies concluded that, at least for these specific Pex1 and Pex7 genetic mutations, there is no discernible difference in the structure of the enamel of these animals.

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Asset Metadata

Creator Bedi, Yudhishtar Singh (author)

Core Title Characterization of mouse models of peroxisome biogenesis disorders: a study of dental enamel phenotypes

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 10/11/2013

Defense Date 09/06/2013

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

Tag enamel defects,mouse model,OAI-PMH Harvest,peroxisome biogenesis disorders,PEX1,Pex7

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hacia, Joseph G. (committee chair), Kalra, Vijay K. (committee chair), Paine, Michael (committee chair), Tokes, Zoltan A. (committee member)

Creator Email ybedi@usc.edu,yudibedi@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-337461

Unique identifier UC11296373

Identifier etd-BediYudhis-2095.pdf (filename),usctheses-c3-337461 (legacy record id)

Legacy Identifier etd-BediYudhis-2095.pdf

Dmrecord 337461

Document Type Thesis

Format application/pdf (imt)

Rights Bedi, Yudhishtar Singh

Type texts

Source 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 a...

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