behavioral assesment of the pahenu2 mouse...

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BEHAVIORAL ASSESMENT OF THE Pahenu2 MOUSE MODEL OF PHENYLKETONURIA

By

PADMINI ASHOK KUMAR

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

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2011

© 2011 Padmini Ashok Kumar

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To both my parents for giving me the best education I could ask for

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ACKNOWLEDGMENTS

It is a great privilege for me to offer my heartfelt gratitude to my mentor, Dr. Philip

Laipis for all the encouragement, guidance and inspiration he has provided at every

step for the completion of my master’s thesis. I would like to thank you for giving me the

opportunity to work in your lab.

I would like to extend my sincere thanks to all members of my master’s committee,

Dr. Mark Lewis, Dr. Ammon Peck and Dr. William Zeile for their untiring effort to advice

and help me through the course of this work.

I am deeply grateful to Dr. Mark Lewis for his valuable suggestions and timely

help. I would like to thank Sasha Vaziri for taking the time out to help me get acquainted

with Observer software. I would also like to thank the Lewis lab members who have

been extremely friendly and supportive throughout the completion of my project.

I would like to acknowledge all the members of my lab for their untiring help and

support towards the completion of this work.

I appreciate Joyce Conners and all the IDP staff for their continual support towards

the completion of my degree.

I would like to thank my father Dr. J. M. Ashok Kumar for always encouraging me

to believe that I could achieve anything I put my mind to. This is for you dad.

Above all, I thank God, for providing me His grace and being with me all along the

course of this work.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 INTRODUCTION .................................................................................................... 12

Phenylketonuria ...................................................................................................... 12

History .............................................................................................................. 13

Clinical Features ............................................................................................... 14

Enzymatic phenotype of PKU patients. ...................................................... 14

Biochemical phenotype of PKU patients. ................................................... 15

Phenotype of treated PKU patients ............................................................ 16

Maternal PKU Syndrome .................................................................................. 17

Phenylalanine Metabolic Pathway .......................................................................... 18

2 THE PAHENU2 MOUSE MODEL & PAL TREATMENT ............................................ 21

Animal Model Of PKU ............................................................................................. 21

Alternative Therapies .............................................................................................. 22

Previous Work Done ............................................................................................... 24

PAL Treatment ........................................................................................................ 25

Behavioral Profile of Pahenu2 Mice ........................................................................... 28

3 EXPERIMENTAL APPROACH ............................................................................... 33

Specific Aims .......................................................................................................... 33

Methods and Materials ............................................................................................ 34

Subjects ............................................................................................................ 34

Behavioral Testing ............................................................................................ 34

Experimental setup .................................................................................... 34

Spatial novelty test ..................................................................................... 35

Previous Recordings ............................................................................................... 35

The Observer .......................................................................................................... 35

Activity .............................................................................................................. 36

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Inactivity ........................................................................................................... 36

Rearing ............................................................................................................. 36

Line Cross ........................................................................................................ 36

Grooming .......................................................................................................... 36

Statistical Analysis .................................................................................................. 37

4 RESULTS FROM THE SPATIAL NOVELTY TEST ................................................ 41

Study 1- Genotype Comparisons ............................................................................ 41

Study 2- Treatment With Pal ................................................................................... 43

5 DISCUSSION AND FUTURE DIRECTIONS .......................................................... 68

Discussion .............................................................................................................. 68

Future Directions .................................................................................................... 70

LIST OF REFERENCES ............................................................................................... 73

BIOGRAPHICAL SKETCH ............................................................................................ 78

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LIST OF TABLES

Table page 4-1 Behavioral Analysis-STUDY ............................................................................... 44

4-2 Tukey’s studentized range test results for full rear- p=0.0003, F=10.09.. ........... 50

4-3 Tukey’s studentized range test results for half rear-p=0.0001, F=15.38.. ........... 52

4-4 Tukey’s studentized range test results for center rear- p=0.0450, F=3.38.. ........ 54

4-5 Tukey’s studentized range test results for line cross- p<0.0001; F=15.58.. ........ 57

4-6 Behavioral Analysis- STUDY 2 (PKU mice). ....................................................... 60

4-7 Activity Data Analysis- STUDY 2 p=0.734. ......................................................... 61

4-8 Inactivity Data Analysis- STUDY 2 p=0.770. ...................................................... 62

4-9 Full Rear Data Analysis- STUDY 2 p=0.076 ....................................................... 64

4-10 Half Rear Data Analysis- STUDY 2 p=0.334. ..................................................... 65

4-11 Line Cross Data Analysis- STUDY 2 p=0.164 .................................................... 67

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LIST OF FIGURES

Figure page 1-1 Conversion of Phenylalanine to Tyrosine by PAH.. ............................................ 20

2-1 Comparison of the enzymatic pathway of PAH and PAL. ................................... 32

3-1 Observational chamber. ..................................................................................... 38

3-2 Synctactic grooming pattern exhibited by mice................................................... 39

3-3 Box plot description. ........................................................................................... 40

4-1 Distribution of activity and inactivity data for STUDY 1(WT, HET and PKU) in terms of total percentage of time. ....................................................................... 45

4-2 Box plot depicting distribution of activity data for STUDY 1.. .............................. 46

4-3 Box plot depicting distribution of inactivity data for STUDY 1 (p=0.4115).. ......... 47

4-4 Distribution of rearing data for STUDY 1 (WT, HET and PKU) in terms of rate per minute........................................................................................................... 48

4-5 Box plot depicting distribution of full rear data for STUDY 1 (p=0.4115)............. 49

4-6 Box plot depicting distribution of half rear data for STUDY 1(p=0.0001). ......... 51

4-7 Box plot depicting distribution of center rear data for STUDY 1 (p=0.0450).. ..... 53

4-8 Distribution of line cross data for STUDY 1 (WT, HET and PKU) in terms of rate per minute. .................................................................................................. 55

4-9 Box plot depicting distribution of line cross data for STUDY 1 (p<.0001).. ......... 56

4-10 Distribution of grooming data for STUDY 1.. ...................................................... 58

4-11 Distribution of activity and inactivity data for STUDY 2 (on and off treatment) in terms of percentage of time.. .......................................................................... 60

4-12 Distribution of rearing data for STUDY 2 (on and off treatment) in terms of rate per minute ................................................................................................... 63

4-13 Distribution of line cross data for STUDY 2 (on and off treatment) in terms of rate per minute ................................................................................................... 66

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LIST OF ABBREVIATIONS

6-PTS 6- pyruvoyl tetrahydropterin

AMPA (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BBB Blood Brain Barrier

BH4 Tetrahydrobiopterin

BH4 Tetrahydrobiopterin

cAMP cyclic Adenosine Monophosphate

CHD Congenital Heart Disease

DHPR Dihydropteridine reductase

GTP-CH Guanosine triphosphate cyclohydrolase

HET Heterozygous for the Pahenu2 mutation at the PAH locus

HPA Hyperphenylalaninemia

HPA Hyperphenylalaninemia

IEM Inborn Errors of Metabolism

IQR Interquartile Range

LNAA Large Neutral Amino Acids

MPKU Maternal Phenylketonuria

NMDA N-methyl-D-aspartate

PAH Phenylalanine Hydroxylase

PAL Phenylalanine ammonia lyase

Phe Phenylalanine

PKU Homozygous for the Pahenu2 mutation at the PAH locus i.e. exhibits symptoms of phenylketonuria.

PKU Phenylketonuria

WT Wild Type

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

BEHAVIORAL ASSESMENT OF THE Pahenu2 MOUSE MODEL OF

PHENYLKETONURIA

By

Padmini Ashok Kumar

May 2011

Chair: Philip J. Laipis Major: Medical Sciences

Phenylketonuria (PKU) is an autosomal recessive disease caused as result of an

inborn error in the metabolism of the essential amino acid phenylalanine (Phe). It is

characterized by an elevation of phenylalanine in the blood, brain and other major

organs. The most common symptom is severe mental retardation. Most PKU patients

have mutations in the phenylalanine hydroxylase (PAH) gene locus resulting in deficient

PAH activity. A Phe restricted diet supplemented with other essential amino acids is the

current standard of treatment. Unfortunately this diet is both expensive and unpleasant.

The adult PKU patient off diet is subject to a number of serious cognitive deficits. There

has also been an increase in the incidence of birth defects in children born to PKU

mothers (maternal PKU syndrome).

Enzyme substitution therapy with a recombinant form of a cyanobacterial enzyme,

Phenylalanine ammonia lyase (PAL) is being explored as an alternative therapy to

dietary treatment. This enzyme requires no cofactors to aid in phenylalanine

metabolism, has no embryotoxic effects and is stable under a wide temperature range.

The work described in this thesis examined the effect of this enzyme substitution

therapy on behavioral characteristics of the Pahenu2 mice.

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Two behavioral studies were undertaken to study the cognitive deficits

experienced by PKU patients in a mouse model. Typical parameters of mouse behavior

such as activity, line cross, rearing and grooming, were measured. The first study

explored the behavioral differences between mouse genotypes (wild type, heterozygote,

and PKU). Rearing data matched that obtained from previous studies, however exact

replication of activity and grooming data was not found. The second study investigated

behavioral response to treatment of severe PKU with PAL. Data obtained from these

observations showed improvement in rearing and small movements (line cross) in

Pahenu2 mice while on treatment, and a significant decline in improvement once taken off

treatment. These observations will be useful in designing studies of PKU mice to

determine behavioral changes associated with PAL treatment while also adding to the

growing body of evidence that PAL treatment of human PKU patients will be a valuable

addition to the existing dietary therapy.

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CHAPTER 1 INTRODUCTION

Phenylketonuria

Phenylketonuria (PKU) is one of the most commonly inherited genetic disorders,

affecting approximately 1 child in every 25000 live births.1 It is an autosomal recessive

disorder that results in a buildup of phenylalanine in the blood, brain and other major

organs. Phenylalanine hydroxylase is essential for the successful catabolism of

phenylalanine into tyrosine thereby eliminating a buildup of excessive Phe in the

circulation. More than 97% of PKU patients possess mutations in the PAH gene locus

resulting in deficient phenylalanine hydroxylase (PAH) activity. However around 2% of

the PKU patient population is instead deficient in tetrahydrobiopterin (BH4) biosynthesis

or recycling (malignant PKU) resulting indirectly in decreased PAH activity and

hyperphenylalaninemia (HPA). 2 PKU is characterized by severe mental retardation,

seizures, and other complications.

Early detection of PKU (plasma Phe levels above1mM) is required in order to

prevent the phenotypic manifestations of this disease. Treatment with a Phe restricted

diet is usually started within the first few weeks of birth and is recommended to be

continued for life in order to maintain a non-toxic range of Phe. Strict adherence to this

diet will result in near normal brain and cognitive development however the treatment is

unpalatable and expensive. Another increasing problem is maternal PKU, where PKU

mothers fail to adhere to this stringent diet during pregnancy. This results in elevated

plasma Phe levels that are detrimental to the fetus. This chapter discusses the current

knowledge on phenylketonuria, previous work done, and the complications arising from

maternal PKU.

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History

The term ―inborn error of metabolism‖ (IEM) was coined by Sir Archibald Garrod, a

British physician honored for his pioneering work in the field of metabolic disorders such

as alkaptonuria, cystinuria, pentosuria, and albinism. Early descriptions of the classic

PKU phenotype given by Folling in 1934 indicate that these patients exhibit severe

mental retardation, seizures, eczema, behavioral disturbances and spastic gait.1 When

approached by the mother of two mentally retarded children he performed a series of

biochemical assays and discovered the presence of phenylpyruvic acid in their urine

samples. He then went on to determine if all patients suffering from mental retardation

excreted this specific acid in their urine.1 When Folling discovered that most of his

patients also had siblings who displayed similar characteristics he was quickly able to

deduce that this disease was inherited in a recessive pattern. He had thus established

the first relationship between mental retardation with a known chemical trait.4

In 1937, this disease (previously termed as Folling’s disease) was renamed

phenylketonuria (PKU) by Dr. Lionel Penrose in order to emphasize the importance of

the biochemical trait, presence of phenylpyruvic acid in the urine.3 Further work was

done by Penrose (UK) and Jervis (US) in order to better understand this inborn error of

metabolism and its consequent effect on intelligence. Their studies showed no sex

chromosome linkage confirming that the disease was an autosomal recessive disorder

with unclear phenotype influencing factors, possibly environmental and genetic. They

also observed that this disorder was most prevalent in Caucasian populations with the

gene frequency being 1 in 100 in the US and UK. By presenting PKU as an example,

Penrose was able to challenge the principles of eugenics since proponents of this

science would have had to sterilize around 1% of the population in order to prevent the

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occurrence of the disease.4 He also proposed that by altering the body’s metabolism we

could alter the behavioral abnormalities caused as a result of this disorder, accurately

predicting the future direction of PKU treatment options.

Work done by Jervis in 1947 showed that patients with Phe levels above normal

physiological levels (>0.12mM but < 1mM) exhibited hyperphenylalaninemia (HPA) that

if left untreated could develop into PKU. In 1953 Udenfriend and Cooper showed that

the deficient enzyme involved in this disease was liver PAH. Bickel was the first to

propose reducing Phe intake as treatment to alleviate the psychiatric manifestations of

this disease. He achieved this by using a Phe-restricted casein hydrolysate.5 The need

to identify PKU patients at an early stage became clearly apparent in order to prevent

the severe mental retardation that would afflict these children otherwise. The first ferric

chloride diaper test was performed in 1957 but was soon found to produce ambiguous

results in newborns. In the early 1960s more accurate assays were developed that

involved screening blood samples for elevated Phe levels using bacterial cultures that

were dependent on Phe for growth. This made neonatal detection and treatment with a

Phe restricted diet possible. Since then regular PKU testing has been implemented in

many countries giving thousands of PKU patients the chance to develop normally.6

Clinical Features

Enzymatic phenotype of PKU patients.

Patients with phenylketonuria (PKU) usually express a PAH-deficient phenotype.

Phenylalanine hydroxylase activity in humans is found to be present only in hepatic and

renal tissue. In vitro analysis of PAH activity in PKU patients has often shown less than

1% of normal activity. In cases of non PKU hyperphenylalaninemia PAH activity is

approximately 1% that of normal activity. Patients who exhibit defects in BH4 synthesis,

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i.e. guanosine triphosphate cyclohydrolase (GTP-CH) deficiency, have less than 4%

normal activity in liver biopsy material.7 Patients who are 6-pyruvoyl tetrahydropterin (6-

PTS) deficient have varying levels of severity. The ―typical’ form of 6-PTS deficiency

that affects brain, splanchnic and erythrocyte enzyme activity exhibits 0-20% of normal

erythrocyte activity; the ―partial‖ form exhibits erythrocyte enzyme activity ranges

starting from as low as 5% but has enough brain 6-PTS activity to maintain BH4

synthesis .

Biochemical phenotype of PKU patients.

Extensive work done previously in mouse models has provided us with sufficient

data to elucidate the biochemical phenotype of PKU patients. It has been observed that

PKU patients have high Phe levels in the brain. Phenylalanine and other large neutral

amino acids (LNAA) are transported across the blood brain barrier (BBB) via the L type

amino acid transporter. In cases of PKU and hyperphenylalaninemia (HPA) high

phenylalanine concentrations in plasma compete with other large neutral amino acids

(LNAA) for the L type amino acid transporter suppressing their influx into the brain.8

Synthesis of catecholamine and indolamine neurotransmitters such as dopamine and

norepinephrine may be reduced due to lower levels of the essential amino acids

tyrosine and tryptophan in the brain as well as by competition for enzyme active sites by

phenylalanine.8, 9 It is thought that deficient synthesis of these neurotransmitters is

partially responsible for the neuropsychiatric abnormalities and cognitive deficiencies

seen in PKU patients.8, 10 This observation emphasizes the need for supplementation of

these amino acids in the diet in order to support proper brain function.

A significant decrease in the expression of mono-amine neurotransmitters such as

serotonin, dopamine and norepinephrine has been observed in the prefrontal cortex,

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cingulated cortex, nucleus accumbens and nigrostriatal region of the brain. These

findings have been consistent with a marked decrease in tryptophan hydroxylase,

necessary for the conversion of tryptophan to serotonin, and tyrosine hydroxylase,

necessary for the conversion of tyrosine to dopamine.11

Anomalies in the glutatmatergic system may also be contributing factors to the

pathophysiology of cognitive deficits and abnormalities in brain function observed in

PKU patients. Glutamate, an excitatory neurotransmitter in the central nervous system,

plays a significant role in neuronal physiology by activating glutamate receptors such as

N-methyl-D-aspartate (NMDA) and (RS)-amino-3-hydroxy-5-methyl-4-

isoxazolepropioinic acid (AMPA). Glutamate receptors are responsible for the formation

of synapses during early development. Increased levels of phenylalanine depress

glutamate synaptic transmission, limit neurotransmitter release and compete for

glutamate binding sites on NMDA and AMPA receptors.12, 2 Increased expression of

NR2A subunit of the NMDA receptor as compared to the NR2B subunit may represent a

premature aging of the PKU brain. It has been observed that the ratio of NR2B/NR2A

expression decreases with age. This net anti-glutamatergic effect of phenylalanine

could be the part of the reason for the cognitive difficulties associated with these

patients.2

Abnormal myelin production has also been observed both in the brains of human

PKU patients and in the PKU mouse brain. Increased Phe levels inhibit ATP-sulfurylase

thereby causing decreased synthesis of sulfatides and an unstable myelin structure.8, 13

Phenotype of treated PKU patients

Early detection of PKU (>1mM) and hyperphenylalaninemia (HPA) (>0.12mM but

< 1mM) has been carried out since the early 1960s.Soon after neonatal detection of

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hyperphenylalaninemia, patients were placed on a stringent Phe restricted diet. This

diet aimed at providing base levels of phenylalanine just sufficient for normal protein

synthesis, but too low to reach abnormal levels in order to prevent onset of the

characteristic neuropsychological phenotype of PKU. The diet comprises of a mixture of

free amino acids and protein hydrolysates and is diluted in water before consumption. 14

The quality of these products has greatly improved since the 1960s in terms of

nutritional content however the diet still remains unpalatable. Young children and

adolescents suffering from PKU and hyperphenylalaninemia must be closely supervised

in order to ensure proper compliance with the diet.

Patients must maintain this diet for life as termination or even slight relaxation of

the diet may lead to a loss of IQ. Previous studies have shown that PKU patients who

had poor dietary control experienced a drop in IQ and behavioral abnormalities as

compared to well controlled PKU patients who maintained near normal Phe levels. 15

Many patients however find it extremely difficult to maintain these strict dietary

requirements and end up terminating the treatment prematurely. This emphasizes the

need for alternative treatment options.

Maternal PKU Syndrome

Maternal PKU syndrome is a teratogenic syndrome caused when elevated Phe

levels in a pregnant PKU mother adversely affects her fetus. Maternal PKU has been

shown to cause fetal brain damage as well as growth and cardiac abnormalities.

Contra-intuitively the success of early detection and treatment of female PKU patients is

a contributing factor to the increased incidence of this condition. Women who have been

successfully treated for HPA and PKU enter their reproductive years as healthy

individuals capable of bearing children.16 However if during pregnancy they fail to

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control their Phe levels, they may cause severe mental retardation and other

abnormalities in the fetus.17 Diagnosis of maternal PKU (MPKU) in known PKU mothers

involves inferences made from ultrasound readings, close observations of fetal

development and close monitoring of growth and cognitive development after birth.18

Studies have shown normal development for up to two weeks after birth when PKU

mothers were placed on a stringent diet with Phe levels maintained at around 0.16-0.44

mM. The most effective treatment for maternal PKU is prevention. Failure to strictly

maintain normal Phe levels will have deleterious effects on neuronal multiplication and

myelogenesis. The common complications arising from MPKU are growth defects,

microcephaly, congenital heart defects (CHD), and mental retardation. Young girls who

are diagnosed with PKU or mild hyperphenylalaninemia should be counseled on the

importance of strict diet adherence and the complications of unplanned pregnancies.18

PAH deficiency in children of PKU females is dependent on the father’s genetic

makeup. If the father has PKU the child will definitely be PKU. If he has two normal PAH

genes then the child will be heterozygous; if he is heterozygous (i.e. a PKU carrier) then

the child will have a 50% probability of being PKU.19

Phenylalanine Metabolic Pathway

Phenylalanine is an essential amino acid; after ingestion it is converted into

protein (25%) and tyrosine (75%).20 Its catabolism is fairly complex as tyrosine is also

involved in the synthesis of neurotransmitters dopamine, epinephrine and

norepinephrine. In PKU patients conversion of phenylalanine to protein is the only way

to eliminate excess phenylalanine in the body as conversion to tyrosine and its further

catabolism is impossible.20 The critical enzyme involved in the breakdown of

phenylalanine is phenylalanine hydroxylase (PAH) (Figure 1-1). Phenylalanine

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hyroxylase is confined to the hepatic and renal tissues although phenylalanine is utilized

by all cells for protein synthesis. It is a tetrameric enzyme and is a homopolymer. It is a

substrate for cyclic adenosine-3’,5’-monophosphate (cAMP)-dependent protein kinase

and is a metalloprotein requiring 1mol of iron per mol of subunit. BH4 is an essential

cofactor required for the hydroxylation of phenylalanine to tyrosine by PAH. After the

hydroxylation reaction BH4 is converted to pterin-4a-carbinolamine. Dihydropteridine

reductase (DHPR) regenerates BH4 from quinone-dihydrobiopterin (q BH2), produced

from the recycling of pterin-4a-cabinolamine, and NADH ; a de novo supply of BH4 is

maintained by means of a pathway that is catalyzed by GTP-CH, 6-PTS and sepiapterin

reductase.7

The most basic requirements for this hydroxylation reaction are phenylalanine

hydroxylase, molecular oxygen, the amino acid substrate (phenylalanine) and

tetrahydrobiopterin (BH4). PAH is activated by phenylalanine and also by

phosphorylation by cAMP-dependent protein.21 Blood glucagon levels indirectly affect

the rate at which Phe is cleared from the system. After a meal cAMP levels increase

thereby activating PAH. BH4 maintains PAH in a mildly active conformation until further

activated by Phe. PAH has two major domains of interest at the carboxy and amino

terminals respectively. The segment at the carboxy terminal is highly conserved and

involves interaction with BH4; the one at the amino terminal is not as conserved and is

involved in substrate specificity, phosphorylation and catalytic activity.21 The majority of

mutations occurring in the PAH gene locus are either missense or deletion mutations.

The mutant enzyme synthesized retains partial function but altered stability and catalytic

rates, thereby resulting in an increase in the turnover of the protein.

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PHENYLALANINE METABOLIC PATHWAY

Figure 1-1. Conversion of Phenylalanine to Tyrosine by PAH.Tyrosine is further

converted into L-Dopa, other essential neurotransmitters, fumarate, and acetoacetate. Deficient PAH activity results in a buildup of phenylalanine which is then slowly converted to phenylketones that can be detected in the urine of PKU patients.

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CHAPTER 2 THE PAHENU2 MOUSE MODEL & PAL TREATMENT

Animal Model Of PKU

The Pahenu2 mouse model of PKU was developed in response to a pressing need

to develop a genetic mouse model closely resembling human PKU. This mouse model

of PKU has been used extensively in studies to determine the underlying

neuropathogenesis of this disease and possible treatment modalities. Initially, rat

models of PKU were developed by introducing Phe analogs to inhibit PAH activity but

various side effects arising from this treatment prevented the use of these rodent

models in further studies. The Pahenu1 mouse model created by McDonald and

colleagues61 failed to duplicate the biological effects seen in human PKU. Further

mutational analysis was performed by mating male BTBR mice, treated with

ethylnitrosourea (ENU) a germ line mutagen, to female Pahenu1 mice. This revealed two

new mutant alleles in the PAH locus and from each, separate congenic inbred mutant

BTBR lines were established. These were the Pahenu2 and Pahenu3 mouse models of

which the former showed greater resemblance to the biochemical characteristics of

human PKU.27, 28

The Pahenu2 mutant has a phenotype closely resembling human PKU with

significantly elevated Phe levels and deficient PAH activity. These mice are smaller

compared to the WT BTBR and HET Pahenu2 mice and have a lighter fur coat reflecting

the inhibition of melanin biosynthesis. A striking similarity in these mice to human PKU

patients was the marked hypopigmentation observed at around 2 weeks of age that

persists for life. Serum Phe levels in these PKU mice were shown to be almost 20 to 30

times that of their wild type counterparts. Comparison studies were carried out between

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homozygous PKU mice and control heterozygotes that were produced by crossing

carrier females with male PKU mutant mice. The results from these studies showed

grooming abnormalities, impaired motor function and cognitive deficits in the PKU

mutant mice as compared to the heterozygotes.

Pahenu2 mice also serve as good models for maternal PKU syndrome. The female

Pahenu2 mice had near normal size litters, however, most pups did not survive beyond

the first few hours after birth. As in humans this effect depends largely on the genotype

of the mother.28 When placed on a Phe restricted diet these animals showed a

significant decrease in serum Phe levels as well as reduced ketone concentrations in

their urine. The survival rate of pups born to Pahenu2 female mice placed on the

controlled diet was significantly greater than those on a standard diet. Reversal of

classic PKU biochemical characteristics exhibited by these mice upon implementation of

the Phe restricted diet is observed. A dramatic increase in the incidence of pup survival

was observed in the female Pahenu2 mice as a result of restricted Phe intake.28

Analyses of PKU mice treated with gene therapy carried out by Embury25, have

shown reversal of the neuropathologic changes associated with this disease. She

determined that disturbances in monoamine metabolism could affect the morphology of

the nigrostriatal regions of the mouse brain. Results from her experiments showed

reduced monoamine metabolite levels and decreased dopaminergic cells in the

substantia niagra regions of PKU mice.25

Alternative Therapies

The inability of certain patients to adhere to the expensive and unpleasant Phe

restricted diet and the rising number of maternal PKU cases has made the need for

alternative therapies abundantly clear.

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BH4 supplementation has been attempted in patients who do not have classic PKU

but possess mutations that are responsive to the cofactor supplementation. This

treatment is patient gene specific; BH4 supplementation helps prevent misfolding and

inactivation of mutant missense proteins. Various trials have shown normal

development and controlled Phe levels in patients treated with BH4 supplementation.

However, further studies are yet to be performed to assess safety of this treatment for

maternal PKU. BH4 supplementation is more expensive than the regular Phe restricted

diet, however, it could prevent day time peaks of Phe levels in PKU mothers.22

Enzyme replacement therapy with phenylalanine ammonia lyase (PAL) could be

an interesting alternative to a Phe restricted diet. Oral administration of enteral gelatin

coated PAL capsules have been shown to reduce Phe levels up to 22% in PKU

patients. PAL modified with polyethylene glycol (PEG) was also employed and studies

in the Pahenu2 mouse model have shown that this form of the enzyme has increased

blood circulating time.23, 24

Gene therapy would be an ideal form of treatment to treat PKU patients and

prevent maternal PKU syndrome. Previous work has shown that this may not yet be the

most appropriate method of treatment. With the help of recombinant adenovirus

decreased Phe levels have been achieved in the BTBR Pahenu2 mouse model

employing Rous Sarcoma virus LTR and CAG promoters respectively. Human PAH was

utilized in these trials as opposed to mouse PAH. The complications that arose with

these trials however was that antibodies were raised against the adenovirus and

complete reversal of the outcomes of the treatment were observed in two weeks.20, 25

24

Previous Work Done

Work done previously in the Laipis lab focused on a histological assessment of the

Pahenu2 mouse model of PKU to better understand the neuropathogenesis of the

disease, AAV-mediated gene therapy and enzyme substitution therapy using PAL.

Immunohistochemical studies of dopaminergic regions were conducted by

Embury26 to achieve a better understanding of the neuropathologic processes

associated with this disease. The substantia niagra (SN) and hypothalamus showed

increased cellularity due to infiltration by CD11b macrophages. Increased expression of

inducible nitric oxide synthase was also observed by these CD11b macrophages.

Expression of i-NOS by CD11b positive cells could also be a compensatory response to

reduce dopamine release in conditions of oxidative stress.26 A striking abnormality

observed was the cytoplasmic vacuolar degeneration of dopaminergic neuronal cell

bodies. Infiltration of dopaminergic regions by cd11b macrophages was also noted.

Reduced monoamine metabolite levels in the striatal regions was measured in brain

tissue from PKU mice. Results from her experiments showed decreased dopaminergic

cells in the substantia niagra regions of PKU mice. Expression of nestin-glial fibrillary

acid protein (GFAP) as a regenerative response to nigrostriatal degeneration was seen.

Portal vein administration of gene therapy vectors (rAAV-mPAH) to Pahenu2 mice

showed significant reduction in serum Phe levels and reversal of the neurodegenerative

changes describe previously.25 As a result of controlled Phe levels oxidative damage

was halted, dopaminergic neurons in the SN regained normal morphology and the

observed infiltration by cd11b macrophages was reversed. These results show that the

neurodegenerative changes caused as a result of increased Phe levels are indeed

reversible with gene therapy. Thus, PKU mice could be successfully treated although

25

the large amount of vector required makes human application difficult. Gene therapy

approaches in humans has not yet been attempted. Thus alternative methods of

treatment are being explored.

PAL Treatment

The most widely used treatment option for PKU is the administration of a Phe

restricted diet that has been implemented since the early 1950s. Strict compliance with

this diet is required in order to prevent the onset of cognitive impairments associated

with this disease. Unfortunately most adult PKU patients find it difficult to strictly adhere

to this treatment option which is both expensive and unpleasant. Over the past few

years this dietary supplement has been refined but still lacks certain essential nutrients

that could affect neural development. Alternative therapies for PKU treatment are

currently being explored in an attempt to overcome the complications associated with

this dietary supplementation.

Somatic gene therapy for therapeutic liver repopulation is one of the treatment

options currently being explored as an alternative to the Phe restricted diet, however

work done in this area is still in experimental stages.33, 34 Dietary supplementation of

large neutral amino acids has also being considered as an alternative option in the

hopes of out-competing Phe for transport across the blood brain barrier. This is only

recommended for patients who find it extremely difficult to comply with a Phe restricted

diet.35, 36 Liver transplants are another viable clinical option in extremely rare cases.37

PAH cofactor supplementation is an option for patients who have PAH missense

mutations. BH4 responsiveness has been shown to lower Phe levels by around 60% in

patients who exhibit mild hyperphenylalaninemia.38 This treatment option is not effective

26

in patients who have a more severe form of classical PKU. This group of non-

responsive PKU patients could benefit from enzyme replacement or gene therapy.

In enzyme therapy, PKU patients can either be treated for deficient PAH activity by

supplementation with phenylalanine hydroxylase or phenylalanine ammonia lyase, a

plant enzyme capable of degrading phenylalanine. There are several challenges

associated with phenylalanine hydroxylase enzyme supplementation. PAH is

intrinsically unstable making its large scale isolation extremely difficult. In order to

maintain complete catalytic hydroxylating activity of the enzyme it must be isolated as

an intact multi-enzyme complex.39 Attempts to develop modified forms of PAH that

retain catalytic activity and enhance stability have been reported. Attempts at reducing

the immunogenic response to this enzyme have been made by modifying it through

chemical conjugation to polyethylene glycol (PEG) .40 The requirement of the essential

cofactor BH4 along with its potential to illicit an immunogenic response complicate its

usage as a viable therapeutic option.

Phenylalanine ammonia lyase is a plant, fungal, and lower eukaryotic organism

enzyme capable of breaking down L-phenylalanine to trans-cinnamic acid and ammonia

(Figure 2-1); this catabolic pathway does not exist in animals However, the products of

Phe break down are innocuous in mammals. Trans-cinnamic acid is then excreted as

hippurate in urine41 along with small amounts of cinnamic and benzoic acid; ammonia is

metabolized by the urea pathway. PAL is an autocatalytic enzyme and experiments

carried out on laboratory animals have revealed no embryotoxic effects.42, 43, 44 Enzyme

replacement therapy with PAL is more cost effective than dietary therapy as a result of

the abundance of recombinant PAL (purified from a bacterial source). The development

27

of various mouse models of PKU has greatly aided the progress of clinical investigation

.45, 30 PAL was found to retain activity at a pH of 8.5 and temperature of 30˚C 47making it

suitable for an enteral route of PAL therapy however it will be degraded in the intestinal

lumen unless protected. Further modifications are required to prevent proteolytic

degradation of PAL.48 Studies involving administration of PAL in an enteric-coated

gelatin capsule were carried out and results from these studies showed reduced Phe

levels in the blood of PKU patients by 22%.49 Only 20% of enzyme activity was retained

when encapsulated PAL was orally administered.50 To overcome this reduced enzyme

activity attempts were made at entrapment in silk fibroin, however, no clear results exist

to prove its effectiveness against gastric acidity.51 Recent studies involve the oral

administration of recombinant PAL, in its original Escherichia coli expression cells, to

Pahenu2 mice.52 Phe levels were shown to be reduced by 31% in one hour after

administration and 44% after two hours. However low levels of specificity and

inefficiency at pH 7 are complications that need to be overcome with this treatment.

Intraperitoneal injections with recombinant PAL were shown to lower plasma Phe levels

significantly for up to 24 hours after administration53, but this is a clinically complicated

delivery route unsuitable for routine, patient-administered use.

The Laipis laboratory has investigated subcutaneous administration of a modified

form of PAL. Initially, subcutaneous administration of PAL was found to illicit a severe

immunogenic reaction leading to a short half life of the enzyme in the circulation.54

Further modification of the enzyme is required in order to inhibit immunogenicity. An

extracorporeal multitubular enzyme reactor supplied with immobilized PAL was used to

reduce Phe levels without entering the circulation and was found to reduce Phe levels

28

by 77%.55,56 However, this is not feasible as a long term treatment option but maybe

used for management of Phe levels in pregnant women. In order to reduce the

immunoreactions associated with subcutaneous PAL administration, PEGylation of PAL

was attempted. Initial injections with PEGylated PAL showed an increased half life of

the enzyme but the enzyme was still rapidly cleared from the circulation after multiple

injections.57 Various studies were carried out to chemically modify recombinant PAL in

attempts to retain specificity and reduce immunogenicity. Conjugation of recombinant

PAL with branched and linear polyethylene glycols have shown promising results for the

future treatment of PKU patients with subcutaneous recombinant PAL enzyme

substitution.58, 59 Phase I clinical trials have been completed and Phase II clinical trials

are underway.61

Behavioral Profile of Pahenu2 Mice

Prior studies have elucidated the biochemical characteristics and metabolic

pathways that are implicated with the manifestation of this disease. However a better

understanding of the causative factors underlying the psychiatric manifestations of this

disease is yet to be determined. Relatively few observational studies have been

performed to identify any cognitive deficits or significant behavioral patterns exhibited by

PKU mice. Previous behavioral studies done on the Pahenu2 mouse model of PKU will

be discussed here. Behavioral analysis to detect cognitive deficits in mice involves

observing changes in characteristics such as latent learning, object recognition, spatial

novelty and short term memory.

A series of comparative reversal tests were performed by Tang et al.29 to observe

any behavioral differences between the Pahenu2 and control mice (BTBR wild type or

heterozygous mice). The first task involved retrieval of a food reward placed in baited

29

and non baited caps using a specific scent (cinnamon or nutmeg) as a cue for

discrimination. A reversal test was then performed where the scent previously

associated with the non baited cap now contained the food reward. Subsequent re-

reversal tasks were also performed.29 Results from these tests showed no significant

difference between wild type and Pahenu2 mice on the first reversal task however the

Pahenu2 mice were found to be severely impaired on the second reversal task. Latent

learning was also tested in these mice. They were allowed to explore a novel

environment fitted with a water bottle. After being water deprived they were reintroduced

into the same environment and the time taken to find the water bottle was assessed.

These results showed significant impairment in latent learning by the Pahenu2 mice in

comparison to the other two groups.29

Further studies were carried out by Sarkissan and colleagues.30 They employed a

T maze and eight-arm radial maze for their observational tests. The T maze test

involved food acquisition by food deprived mice. It was observed that the Pahenu2 mice

took significantly longer to locate the arm containing the bait.30 In the eight-arm radial

maze test, food rewards were randomly placed in different arms. The baited arms were

identified by means of a light placed directly above the food cup. The mice were then

expected to retrieve all food rewards while minimizing the number of entries into

previously visited arms.30 Observations from this experiment showed that the Pahenu2

mice re-entered previously visited arms more frequently than mice in the control group.

From these observations Sarkissan et al. were able to deduce that Pahenu2 mice showed

significant deficits in areas of simple discrimination, latent learning, habit learning, and

short term memory.30

30

Behavioral tests carried out by Cabib et al.31 present the most compelling

evidence that PKU mice do indeed show signs of significant cognitive deficits. This

study involved the comparison of homozygous PKU and heterozygous (HET) Pahenu2

mice with the BTBR background strain and two inbred strains C57BL/6 and DBA/2.31 A

spatial novelty test was performed to collect data on specific behavioral responses such

as grooming, rearing, inactivity and locomotion. Exploration of novel objects placed

within the observational chamber was assessed. The ability to discriminate between

spatial and non spatial novelty was determined by the time spent by each genotype

exploring displaced and non-displaced objects.31 These experiments required minimal

training and the mice were acclimatized to the observational chamber before any

experiments were run. All observations were recorded with a camera positioned outside

the sound attenuated observation cubicle. The video clips obtained after observations

were then quantified by an experimenter with no prior exposure to treatment conditions.

31 After collection of data a number of one way ANOVAS were run for the various

behavioral responses followed by post hoc pair wise comparison tests to test the effects

of genotype on all factors.

Interpretation of data obtained from these experiments showed decreased

locomotion by PKU mice. PKU mice also engaged in excessive grooming and hence

were less mobile in comparison to other groups. No discrimination between genotypes

was noted for exploration of novel objects however PKU and DBA mice did show

deficits in recognition of displaced objects. New object exploration and emotional

reactivity among the different genotypes was the same.31

31

The object recognition test consisted of a pre-test and test session. In the pre

test session mice were introduced into an observational chamber containing two

identical objects and the exploration time by each genotype was recorded. In the test

session the objects used in the pre-test session were replaced with new ones, one

similar to the previous objects and one novel object.31 Analysis of data obtained from

these studies showed that BTBR and DBA mice exhibited differences in the time spent

exploring the objects whereas no difference was observed in PKU and C57 mice. Thus

PKU and C57 showed no discrimination between the novel object and one that had

already been explored.31

Work done by Zagreda et al32 involved odor discrimination and reversal studies

to observe cognitive impairments in the Pahenu2 genetic mouse model of PKU. Their

experiments involved reward retrieval using specific odors as markers to discriminate

between baited and non baited caps. Latent learning was also observed in these mice

with the help of an open field attached to a T-maze.32 On day one the mice were

allowed to explore this novel experimental setup containing water. The mice were then

removed and water deprived for 24 hours before being re-introduced into the same

observational chamber. The results obtained from these experiments showed no

difference with respect to acquisition of food rewards based on odor discrimination

however, female mice exhibited slower reward retrieval (odor discrimination) as

compared to the male mice. 32 The reversal tests revealed impaired odor discrimination

by the Pahenu2 mice as compared to WT and HET groups. There was no difference

between the sexes in performance on the reversal tests. It was observed that the

Pahenu2 mice took significantly longer to finish all reversal tests in comparison to WT and

32

HET groups. The overall percentage of Pahenu2 mice that passed all four reversal trials

was much lower than the other two groups.32 Latent learning results in these mice

showed no difference between sexes. Pahenu2 mice failed to exhibit latent learning,

taking significantly longer to find water on the second day after pre-exposure as

compared to the naïve Pahenu2 mice. The WT and HET mice performed significantly

better than the Pahenu2 mouse. Overall conclusions obtained from these studies showed

that the Pahenu2 mouse exhibited significant cognitive deficits when compared to mice in

the other two groups.32

Figure 2-1. Comparison of the enzymatic pathway of PAH and PAL. (A). Phenylalanine is converted to Tyrosine in a reaction catalyzed by PAH in the presence of the cofactor BH4. Tyrosine is then further catabolized into essential neurotransmitters and other hormones. (B) Phenylalanine is converted to trans-cinnamic acid and ammonia in a reaction catalyzed by PAL that does not require the presence of any additional cofactors. Trans-cinnamic acid is excreted and ammonia is funneled into the urea cycle.

33

CHAPTER 3 EXPERIMENTAL APPROACH

Specific Aims

Specific Aim 1: One of the primary goals of this project was to determine if we

could detect behavioral differences between WT BTBR, HET and PKU mice and also

develop a quantitative measure of any differences.

Specific Aim 2: We wished to determine the effect of PAL treatment on the

behavioral patterns of PKU mice and again quantitatively measure any differences

between treated, untreated and treated animals after removal from treatment.

Much of the work done for this project was aimed at trying to correlate our results

with reported behavioral differences between the three genotypes (WT, HET, PKU)

found in the literature. We wanted to see if the difference in Phe levels between the

three genotypes was reflected in their behavioral patterns. We expected to see high

levels of locomotion (activity) from WT BTBR mice when compared to PKU mice since

motor deficits in the PKU mouse model have been reported in the literature31. We also

expected to see the PKU mice engage in excessive grooming when compared to the

other two genotypes31. PAL treated PKU mice were then observed to quantitate

measurable changes in their behavior during and after treatment. We wanted to

determine if behavioral differences exhibited by these mice could be completely

reversed to mimic that of WT and HET mice once their Phe levels were controlled. We

expected to see improvements in motor activities and rearing during PAL treatment. The

same mice were observed after treatment to determine if the effects of the drug were

reversable.

34

Methods and Materials

Subjects

For the following experiments mice were divided into two studies. Study 1 was

based on genotype and consisted of: 1. Wild type BTBR mice (WT); 2. heterozygous

BTBR mice (HET) derived from crossing wild type mice with Pahenu2 mice and 3.

homozygous Pahenu2 (PKU) mice. Study 2 was based on treatment: 1. PKU mice on

treatment and 2. PKU mice off treatment. This group consisted of PKU mice that were

treated with PAL enzyme supplied by Biomarin Pharmaceuticals Ltd. These mice were

recorded before, during and off PAL treatment. The experimental group was compared

with the PKU, HET and WT animals in Study 1. Observational tests were carried out

during the second half of the light period and all animals were treated in accordance

with IACUC guidelines.

Behavioral Testing

Experimental setup

The apparatus consisted of a circular plexiglass chamber placed over a glass floor

and covered on top by a piece of white cardboard (Figure 3-1). Previous recordings

have shown that a bottom view of the observational chamber is needed for clear

analysis of grooming exhibited by the mice. All sessions were videotaped by means of a

camera positioned below the observational setup. Animals were removed from their

cages and transferred to the recording chamber. Offline analysis of these video clips

was carried out using a computer assisted scoring system (Observer 4.0) to record pre-

defined characteristics that are explained in detail further on in this chapter.

35

Spatial novelty test

Mice from each group were subjected to individual 5 and 10 min test sessions. At

the end of each session the mice were returned to their home cages. All sessions were

recorded and analyzed. In the spatial novelty test mice were introduced into the circular

observational chamber and allowed to explore the novel setting. During these recording

sessions the duration of five behavioral characteristics were observed; activity,

inactivity, rearing, line cross (small movements) and grooming. Grooming was analyzed

only for the WT and PKU mice in the Study 1 and was discontinued for the treatment

group since these mice seemed to groom for very short periods of time and quite

infrequently. At the end of each session mice were carefully removed and placed back

in their home cages. The apparatus was then thoroughly washed and wiped down with

ethanol before the next recording session.

Previous Recordings

Video files of the WT, HET and PKU mice previously recorded were converted

into a format compatible with the The Observer program. This was done employing

123DVD Converter software and video clips of each mouse were then created. Video

recordings of mice in Study 2 (treated with PAL enzyme supplied by Biomarin) were

already in the required format.

The Observer

The behavioral analysis of these animals was carried out with the help of a

computer assisted manual event recorder, The Observer program. After recordings of

all mice had been completed, an experimenter blind to treatment conditions was used to

quantify the behavioral characteristics exhibited by mice in different subject groups.

Before videos were scored using The Observer, the behavioral parameters being

36

observed were clearly defined and reliability was established with another experimenter

(also blind to treatment conditions). For the purpose of quantifying the different

behavioral parameters under analysis the following definitions were associated with

each characteristic.

Activity

The animal was defined as being active if it displaced itself from one point to

another by moving both fore paws and hind limbs.

Inactivity

The animal was defined as being inactive if it remained in the same position

without moving forepaws or hind limbs for more than five seconds.

Rearing

Rearing was divided into three forms: 1.Full rearing: animal would rise up on hind

limbs and support itself against the walls of the observational chamber with both front

paws; 2. Half rear: animal would partially rise up on one hind limb and support itself

against the walls of the observational chamber with a single paw and 3. Center Rear:

animal would rise up on both hind limbs in the center of the observational chamber

without any support.

Line Cross

The observational setup was divided into four quadrants of equal area. When the

head, fore paws and thorax of the mouse crossed one of the lines into another quadrant

it was recorded as one line cross.

Grooming

Grooming was divided into four stages: 1. Paw licks, 2. Single unilateral strokes, 3.

large bilateral strokes, and 4. Body licking (Figure 3-2).

37

Statistical Analysis

Statistical analysis for the data obtained was carried out using SAS and SPSS

statistical software. For the data obtained from Study 1 a number of one way ANOVAs

were run using SAS software to determine the effect of genotype on the different

behavioral parameters. One way ANOVAs enable us to determine the difference in

means between different groups of measurement data. Each behavioral parameter

(measurement variable) was compared between the three different genotypes (WT,

HET and PKU). Box plots were constructed to visualize the variability in measurements

between the different genotypes (Figure 3-3). Tukey’s post hoc pair wise comparison

tests were run after obtaining a significant F value to identify the largest difference in

means between any two given groups. All pair wise comparison tests involve

comparisons between the differences between two means, the critical differences

required for significance and whether 0 is included within the 95% confidence limits to

determine if data distribution is skewed.62 The critical difference required to state

significance is what differentiates the different pair wise comparison tests

The different treatment groups in Study 2 were subjected to paired sample t tests

to determine significant behavioral differences in mice that have been on and off PAL

treatment. For the paired sample t-test the same mice that were administered PAL

treatment were observed off treatment. These set of tests were run with SPSS statistical

software. The output obtained from SPSS shows the value of the t-test with the

associated p-value. This enables us to make decisions about the pair of the sample

mean.

38

Figure 3-1. Observational chamber. The circular, plexiglass chamber was mounted on a wooden stool fitted with a transparent glass floor and the video camera was positioned below. White cardboard covered the top of the circular plexiglass chamber.

39

Figure 3-2. Synctactic grooming pattern exhibited by mice. Phase I- paw licking,

Phase II-single unilateral strokes (paw over single ear), Phase III-Large bilateral strokes (paws over both ears) and Phase IV-Body licking.59

40

MEDIAN

MEAN

25TH PERCENTILE

75TH PERCENTILE

WHISKER

WHISKER

OUTLIER

UPPER INNER FENCE

LOWER INNER FENCE

Figure 3-3. Box plot description. The horizontal line running through the middle of the

rectangle (box) depicts the median. The cross symbol (+) denotes the mean. The ends of the rectangle represent the 75th percentile (upper quartile) and 25th percentile (lower quartile). The difference between the upper quartile and lower quartile is the interquartile range (IQR). The upper and lower inner fences are located at a distance of 3(IQR) from the ends of the box. The whiskers represent maximum and minimum values lying within the inner fences. Any measurement greater than the maximum value depicted by the top whisker or lower than the minimum value depicted by the lower whisker is deemed an outlier and is denoted by an open circle.

41

CHAPTER 4 RESULTS FROM THE SPATIAL NOVELTY TEST

Study 1- Genotype Comparisons

Results obtained from the spatial novelty test show that there was no significant

difference in activity (movement from one position to another by moving both forepaws

and hind limbs) or inactivity (no movement of forepaws or hind limbs for more than 5

sec) between all three genotypes in Study 1 (Figure 4-1) . A noticeable amount of

variability within the PKU data set was observed from the box plots, with the difference

in lengths of the upper and lower whiskers indicating that the data was negatively

skewed (Figure 4-2; Figure 4-3). Since one way ANOVA analysis of the data revealed

insignificant p values (p=0.41, α=0.05 for both activity and in activity) post hoc pair wise

comparison tests were not run. No statistically significant differences in activity or

inactivity were observed between the three genotypes. (Table 4-1).

Rearing was observed as full rears, half rears and center rears (Figure 4-4). Full

rears (animal rises up on hind limbs and supports itself with both front paws) were found

to be exhibited more by HET and WT mice when compared to PKU mice (Figure 4-5).

After one way ANOVA analysis of the data revealed a significant p value (p=0.0003,

α=0.05) post hoc pair wise comparison tests were run. Tukey’s studentized range test

revealed that there were statistically significant differences in full rearing between HET-

PKU and WT-PKU groups but no difference between WT-HET groups (Table 4-2). Half

rears (animal rises up on one hind limb and supports itself with a single paw) were only

exhibited by PKU and HET mice as compared to WT mice that did not engage in any

half rearing (Figure4-6). Results from the one way ANOVA analysis of the data revealed

a significant p value (p=0.0001; α=0.05). Results from Tukey’s studentized range test

42

revealed statistically significant differences between HET-WT and HET-PKU groups but

no differences between WT-PKU groups (Table 4-3).

Center rears (unsupported rearing in the middle of the observational chamber)

were exhibited entirely by WT and HET mice when compared to PKU mice ( Figure 4-

7). After one way ANOVA analysis of the data an adequately low p value was obtained

(p=0.045; α=0.05). Results from Tukey’s studentized range test revealed statistically

significant differences between WT-PKU groups but no significant mean differences

between WT-HET and HET-PKU groups (Table 4-4).

Line cross results for Study 1indicated that WT and HET mice exhibited more line

crosses, i.e. engaged in more smaller movements and hence were overall less

sedentary as compared to the PKU mice (Figure 4-8). Variability in the WT data set was

observed from the box plots with the difference in the lengths of the upper and lower

whiskers indicating the data was positively skewed data (Figure 4-9). After one way

ANOVA analysis of the data, a significant p value was obtained (p=0.0001, α=0.05).

Tukey’s studentized range test showed statistically significant differences between WT-

PKU and HET-PKU groups and no difference between WT-HET groups (Table 4-5).

Grooming was observed in WT and PKU mice. It was seen that both WT and PKU

mice engaged more in paw licking than in other stages of the syntactic grooming pattern

i.e. single unilateral strokes, large bilateral strokes and body licking (Table 4-1). WT and

PKU mice did not exhibit any unilateral strokes and PKU mice did not exhibit any large

bilateral strokes (Figure 4-10). However since the grooming behavior exhibited by these

mice was quite infrequent and for very short periods of time, it was not observed for the

rest of the experimental groups. The difference in grooming patterns from what has

43

previously been reported in mice59 was significant and will be further evaluated in the

Discussion and Future directions chapter.

Study 2- Treatment With Pal

Study 2 examined the question of whether PAL treatment of PKU mice would

change their behavior. There were no differences in activity (movement from one

position to another by moving both forepaws and hind limbs) or inactivity (no movement

of forepaws or hind limbs for more than 5 sec) observed in PKU mice when placed on

treatment or when taken off treatment (Table 4-6; Figure 4-11). A paired sample t test

was run on the data to determine statistically significant differences in activity and

inactivity between on and off drug groups (p=0.734, α=0.05). No statistical significance

for activity or inactivity was observed between on treatment and off treatment groups

(Table 4-7; Table 4-8).

Rearing was observed as full rear, half rear and center rear (Table 4-6). From

the bar graph constructed to show distribution of rearing data both full rearing (animal

rises up on hind limbs and supports itself with both front paws) and half rearing (animal

rises up on one hind limb and supports itself with a single paw), were found to be

noticeably reduced when mice were taken off treatment as opposed to when they were

on treatment (Figure 4-12). However paired sample t test did not reveal statistically

significant mean differences in full rear or half rear between on and off drug groups (Full

rear p=0.076, α=0.05 ; Half rear p=0.334, α=0.05) (Table 4-9).

The distribution of line cross data for mice in Study 2, showed that PKU mice

exhibited more line crosses i.e. more smaller movements and on the whole were less

sedentary when placed on treatment than when taken off the treatment. The bar graph

(Figure 4-13) depicts the distribution of line cross data between the three treatment

44

groups. A paired sample t test however failed to reveal statistically significant

differences in line cross between on and off treatment groups (p=0.164, α=0.05) (Table

4-11).

Table 4-1. Behavioral Analysis-STUDY 1. Data represented below is the average and standard deviation for each experimental group. The mean for activity and inactivity is represented as percentage of time with no noticeable differences between the three genotypes. The mean for line cross is represented as rate per minute with WT and HET engaging in more line crosses when compared with PKU mice. Rearing is also represented as rate per minute. Full rears are exhibited more by WT and HET mice than by PKU. Half rears are exhibited only by HET and PKU mice. Center rears are exhibited only by WT and HET mice. Grooming data was obtained only for WT mice and is represented as percentage of time. WT mice did not engage in frequent grooming as indicated by the above values.

VARIABLES WT(14) HET(8) PKU(17)

MEAN STD DEV MEAN STD DEV MEAN STD DEV

ACTIVE (TIME %) 91.09% 5.78% 91.49% 4.71% 88.68% 6.44%

INACTIVE (TIME %) 8.92% 5.78% 8.51% 4.71% 11.32% 6.44%

LINE CROSS (rate/min) 22.70 7.78 19.45 5.56 11.27 3.72

FULL REAR (rate/min) 4.46 2.39 4.75 2.28 1.77 1.21

HALF REAR (rate/min) 0.01 0.05 0.68 0.50 0.21 0.23

CENTER REAR (rpm) 0.46 0.72 0.33 0.56 0.01 0.02

PAWLICKS (TIME %) 3.52% 5.23% 3.36% 1.94%

LBS (TIME %) 0.34% 0.78% 0.02% 0.04%

HIND LIMBS (TIME %) 0.26% 0.73% 0.40% 0.68%

45

Figure 4-1.Distribution of activity and inactivity data for STUDY 1(WT, HET and PKU) in terms of total percentage of time. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that the average percentage of time spent active (WT x‾=91.09%; HET x‾ =91.49%; PKU x‾ =88.68%) or inactive (WT x‾=8.92%; HET x‾=8.51%; PKU x‾ =11.32%) is similar for all three genotypes. There are no significant differences in activity or inactivity between the three genotypes (WT, HET and PKU) of Study 1.

46

Figure 4-2. Box plot depicting distribution of activity data for STUDY 1. The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are no significant differences in the amount of time being spent active between the three genotypes (HET x‾=91.49%; PKU x‾=88.68%; WT x‾=91.09%). No statistically significant differences in activity between the three genotypes was observed as indicated by the large p-value obtained (p=0.4115, α= 0.05).

47

Figure 4-3. Box plot depicting distribution of inactivity data for STUDY 1 (p=0.4115). The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are no significant differences in the amount of time being spent inactive between the three genotypes (HET x‾=8.51%; PKU x‾=11.32%; WT μ x‾=8.92%). No statistically significant differences in inactivity between the three genotypes was observed as indicated by the large p-value obtained (p=0.4115 , α =0.05).

48

Figure 4-4. Distribution of rearing data for STUDY 1 (WT, HET and PKU) in terms of rate per minute. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that the WT and HET mice engaged in more full rears when compared to PKU mice (WT x‾=4.46; HET x‾=4.75; PKU x‾=1.77). Half rearing was exhibited only by HET and PKU mice (WT x‾=0.01; HET x‾ =0.68; PKU x‾ =0.21). Center rears were exhibited only by WT and HET mice (WT x‾=0.46; HET x‾ =0.33; PKU x‾ =0.01). There are significant differences in rearing between the three different genotypes (WT, HET and PKU) as can be deduced from the above graph.

49

Figure 4-5. Box plot depicting distribution of full rear data for STUDY 1 (p=0.4115). The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are significant differences in the number of full rears between the three genotypes (HET x‾=4.75; PKU x‾=4.46; WT x‾=1.77). Statistically significant differences in half rears between the three genotypes was observed as indicated by the low p-value obtained (p=0.0003, α=0.05).

FULL REAR

50

Table 4-2. Tukey’s studentized range test results for full rear- p=0.0003, F=10.09. The

low p value obtained (p=0.0003, F=10.09) indicates that there is significant difference in full rearing among the three different genotypes in Study 1.The stars in the last column indicate between which genotypes the mean differences are statistically significant. From this table we can determine that there are statistically significant differences in full rearing between HET-PKU and WT-PKU groups however there is no significant difference between HET-WT groups.

COMPARISONS SIGNIFICANT AT THE 0.05 LEVEL ARE INDICATED BY *** GROUP COMPARISON

DIFFERENCE BETWEEN MEANS

SIMULTANEOUS 95% CONFIDENCE LIMITS

HET - WT 0.2886 -1.8020 2.3792 - HET - PKU 2.9750 0.9526 4.9974 *** WT - HET -0.2886 -2.3792 1.8020 - WT - PKU 2.6864 0.9840 4.3888 *** PKU - HET -2.9750 -4.9974 -0.9526 *** PKU - WT -2.6864 -4.3888 -0.9840 ***

51

Figure 4-6. Box plot depicting distribution of half rear data for STUDY 1(p=0.0001). The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are significant differences in the number of half rears between the three genotypes (HET x‾=0.68; PKU x‾=0.21; WT x‾=0.01). Statistically significant differences in half rears between the three genotypes was observed as indicated by the low p-value obtained (p=0.0001 α=0.05).

HALF REAR

52

Table 4-3. Tukey’s studentized range test results for half rear-p=0.0001, F=15.38. The low p value obtained (p=0.0001, F=15.38) indicates that there is significant difference in half rearing among the three different genotypes in Study 1. The stars in the last column indicate between which genotypes the mean differences are statistically significant. From this table we can determine that there are statistically significant differences in full rearing between HET-PKU and HET-WT groups however there is no significant difference between PKU-WT groups.




HET - PKU 0.46574 0.18186 0.74961 *** HET - WT 0.66321 0.36976 0.95666 *** PKU - HET -0.46574 -0.74961 -0.18186 *** PKU - WT 0.19748 -0.04148 0.43644 - WT - HET -0.66321 -0.95666 -0.36976 *** WT - PKU -0.19748 -0.43644 0.04148 -

53

Figure 4-7. Box plot depicting distribution of center rear data for STUDY 1 (p=0.0450). The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are significant differences in the number of center rears between the three genotypes (HET x‾=0.33; PKU x‾=0.01; WT x‾=0.46). Statistically significant differences in center rears between the three genotypes was observed as indicated by the low p-value obtained (p=0.0450, α=0.05).

CENTER REAR

54

Table 4-4. Tukey’s studentized range test results for center rear- p=0.0450, F=3.38. The p value obtained (p=0.0450, F=3.38) is low enough to indicate that there is a significant difference in center rearing among the three different genotypes. The stars in the last column indicate between which genotypes the mean differences are statistically significant. From this table we can determine that there are statistically significant differences in center rearing between WT-PKU groups however there is no significant difference between WT-HET and HET-PKU groups.




WT - HET 0.1330 -0.4048 0.6709 - WT - PKU 0.4540 0.0160 0.8919 *** HET - WT -0.1330 -0.6709 0.4048 - HET - PKU 0.3210 -0.1993 0.8412 - PKU - WT -0.4540 -0.8919 -0.0160 *** PKU - HET -0.3210 -0.8412 0.1993 -

55

Figure 4-8. Distribution of line cross data for STUDY 1 (WT, HET and PKU) in terms of rate per minute. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that the WT and HET mice engaged in more number of line crosses when compared to PKU mice (WT x‾=22.7; HET x‾=19.45; PKU x‾=11.27). This indicates that WT and HET mice exhibited more small movements and were overall less sedentary as compared to the PKU mice. There are significant differences in line cross between the three different genotypes (WT, HET and PKU) as can be seen in the above graph.

56

Figure 4-9. Box plot depicting distribution of line cross data for STUDY 1 (p<.0001).The mean for each group is represented by a diamond inside the rectangle. It can be seen that there are significant differences in the number of line crosses between the three genotypes (HET x‾=19.45; PKU x‾=11.27; WT x‾=22.70). Statistically significant differences in line cross between the three genotypes was observed as indicated by the low p-value obtained (p<.0001).

LINE CROSS

57

Table 4-5. Tukey’s studentized range test results for line cross- p<0.0001; F=15.58. The low p value obtained (p<0.0001; F=15.58) indicates that there is a significant difference in line cross among the three different genotypes in Study 1. The stars in the last column indicate between which genotypes the mean differences are statistically significant. From this table we can determine that there are statistically significant differences in line cross between WT-PKU and HET-PKU groups however there is no significant difference between WT-HET groups.




WT - HET 3.243 -2.001 8.488 - WT - PKU 11.426 7.156 15.697 *** HET - WT -3.243 -8.488 2.001 - HET - PKU 8.183 3.110 13.256 *** PKU - WT -11.426 -15.697 -7.156 *** PKU - HET -8.183 -13.256 -3.110 ***

58

Grooming

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

% Paw Licks % Large Bilateral Strokes % Hind Limbs

Figure 4-10. Distribution of grooming data for STUDY 1. The above figure represents

the distribution of grooming data obtained for WT and PKU mice in Study 1. It can be observed from this graph that WT and PKU mice engage mostly in paw licking and do not follow the syntactic grooming patterns. Both WT and PKU mice do not exhibit any single unilateral strokes. PKU mice do not exhibit any large bilateral strokes when compared to WT mice.

WT PKU

59

Table 4-6. Behavioral Analysis- STUDY 2 (PKU mice). Data represented below is the average for each experimental group. The mean for activity and inactivity is represented as percentage of time with no noticeable differences between the three groups (PRE x‾=77.56%, ON x‾=74.39% or OFF x‾=68.79%). The mean for line cross is represented as rate per minute with mice in pre treatment and on treatment groups engaging in more line crosses when compared with mice off treatment (PRE x‾=9.85, ON x‾=8.68 or OFF x‾=5.81). Rearing is also represented as rate per minute. Full rears are exhibited more by mice pre treatment and on treatment than when taken off treatment (PRE x‾=1.99, ON x‾=1.26 or OFF x‾=0.31). Half rears are exhibited slightly more pre treatment and on treatment when compared to mice off treatment (PRE x‾=0.42, ON x‾=0.47 or OFF x‾=0.26). Center rears are hardly exhibited by PKU mice in Study 2 (PRE x‾=.05, ON x‾=0 or OFF x‾=0).

VARIABLES PRE TREATMENT (22 animals)

ON TREATMENT (20 animals)

OFFTREATMENT (21 animals)

MEAN STDEV MIN

MAX MEAN STDEV MIN (sec)

MAX (sec)

MEAN STDEV MIN (sec)

MAX (sec)

ACTIVE (%) 77.56% 13.85% 9.18

90.98 74.39% 16.54% 6.85 97.99 68.79% 14.06% 10.34 87.63

INACTIVE (%) 22.87% 13.85% 1.69 23.33 25.61% 16..54% 2.0 33.05 31.21% 14.06% 2.78 44.73

LINE CROSS (rate/min)

9.85 3.41 - - 8.68 3.64 - - 5.81 3.5 - -

FULL REAR (rate/min)

1.99 1.24 - - 1.26 1.20 - - 0.31 0.45 - -

HALF REAR (rate/min)

0.42 0.54 - - 0.47 0.76 - - 0.26 0.4 - -

CENTER REAR (rate/min)

0.05 0.17 - - 0 0 - 0 0 0 - -

60

Figure 4-11. Distribution of activity and inactivity data for STUDY 2 (on and off treatment) in terms of percentage of time. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that the average percentage of time spent active (PRE x‾=77.56%; ON x‾ =74.39%; OFF x‾ =68.79%) or inactive (PRE x‾=22.87%; ON x‾ =25.61%; OFF x‾ =31.21%) is similar for all three groups. There are no significant differences in activity or inactivity between the three different treatment groups (pre treatment, on treatment and off treatment) in Study 2.

61

Table 4-7. Activity Data Analysis- STUDY 2 p=0.734. This table shows the results from the student’s t test run for activity data-Study 2. The p value obtained (p=0.734) is greatly over 0.05 (the standard significance level used to run all tests) indicating no significant differences in activity between on or off treatment groups.

PAIRED SAMPLE T-TEST-ACTIVITY

.05918

.05310

.14497

.13008

6

6

.7283

.7000

PAIR1ONDRUG

OFFDRUG

STD ERRORSTD DEVNMEAN

.978.0156PAIR1 ONDRUG

&OFFDRUG

SIGCORLNN

-1.7455 .23122.07893.19333.02833

95% CI OF

DIFFERENCE

LOWER UPPER

STD ERRORSTD DEVIATIONMEAN

.7345.359

Sig.(2 tailed)dft

PAIRED SAMPLES TEST

PAIRED SAMPLES TEST

PAIRED SAMPLES CORRELATIONS

PAIRED SAMPLE STATISTICS

62

Table 4-8. Inactivity Data Analysis- STUDY 2 p=0.770. This table shows the results from the student’s t test run for inactivity data-Study 2. The significance value indicated (p=0.770) is greatly over 0.05 (the standard significance level used to run all tests) indicating no significant differences in inactivity between on or off treatment groups.

PAIRED SAMPLE T-TEST-INACTIVITY

.05566

.05310

.13635

.13008

6

6

.3150

.3000

PAIR1ONDRUG

OFFDRUG



&OFFDRUG

SIGCORLNN

-.10983 .13983.04856.11895.01500

95% CI OF

DIFFERENCE

LOWER UPPER


PAIR1 ONDRUG – OFF

DRUG

.7705 .309

Sig.(2 tailed)dft

PAIRED SAMPLES TEST

PAIRED SAMPLES TEST



63

Figure 4-12. Distribution of rearing data for STUDY 2 (on and off treatment) in terms of

rate per minute. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that mice engaged in more full rears in the pre and on treatment groups when compared to the off treatment group (PRE x‾=1.99; ON x‾=1.26; OFF x‾=0.31). No significant differences in half rearing was observed between the three treatment groups (PRE x‾=0.42; ON x‾ =0.47; OFF x‾ =0.26). Center rears were hardly exhibited by mice in the three different treatment groups (PRE x‾=0.05; ON x‾ =0; OFF x‾ =0). There do seem to be significant differences in full rearing data between the three different groups (PRE, ON and OFF) in Study 2 as can be deduced from the above graph.

64

Table 4-9. Full Rear Data Analysis- STUDY 2 p=0.076.This table shows the results from the student’s t test run for full rear data-Study 2. The significance value indicated (p=0.076) is slightly greater than 0.05 (the standard significance level used to run all tests) indicating that this value is approaching significance however due to the small sample size of the experimental groups in Study 2 statistically significant differences between the two groups (on and off treatment) could not be established.

PAIRED SAMPLE T-TEST-FULL REAR

.48873

.12963

1.19714

.31752

6

6

1.1350

.2283

PAIR1ONDRUG

OFFDRUG



&OFFDRUG

SIGCORLNN

-1.3738 1.95071.40615.99486.90667

95% CI OF DIFFERENCE

LOWER UPPER


.0765 2.232

Sig.(2 tailed)dft

PAIRED SAMPLES TEST

PAIRED SAMPLES TEST



65

Table 4-10. Half Rear Data Analysis- STUDY 2 p=0.334. This table shows the results from the student’s t test run for half rear data-Study 2. The significance value indicated (p=0.334) is greatly over 0.05 (the standard significance level used to run all tests) indicating no significant differences in half rears between on or off treatment groups.

PAIRED SAMPLE T-TEST-HALF REAR

.07940

.11266

.19449

.27595

6

6

.3767

.2450

PAIR1ONDRUG

OFFDRUG



&OFFDRUG

SIGCORLNN

-.18508 .44841.12322.30182.13167

95% CI OF

DIFFERENCE

LOWER UPPER


.334 5 1.069

Sig.(2 tailed)dft

PAIRED SAMPLES TEST

PAIRED SAMPLES TEST



66

Figure 4-13.Distribution of line cross data for STUDY 2 (on and off treatment) in terms of rate per minute. Data represented above is the average and standard error of the mean for each experimental group. It can be seen that mice in pre and on treatment groups engaged in more number of line crosses when compared to the off treatment group (PRE x‾=9.85; ON x‾ =8.68; OFF x‾ =5.81). The above graph indicates a significant difference in line cross in mice when placed on treatment compared to when they were taken off treatment. Mice on treatment exhibited more small movements and were overall less sedentary as compared to the PKU mice.

67

Table 4-11. Line Cross Data Analysis- STUDY 2 p=0.164.This table shows the results from the student’s t test run for line cross data-Study 2. The significance value indicated (p=0.164) is over 0.05 (the standard significance level used to run all tests) however does seem to be approaching significance. Due to the small sample size of mice of the experimental groups in Study 2 statistically significant differences between on and off treatment groups could not be established.

PAIRED SAMPLE T-TEST-LINE CROSS

1.29991

.87628

3.18411

2.14643

6

6

7.633

5.4417

PAIR1ONDRUG

OFFDRUG



&OFFDRUG

SIGCORLNN

-1.26449 5.647821.344503.293352.19167

95% CI OF

DIFFERENCE

LOWER UPPER


.16451.630

Sig.(2 tailed)dft

PAIRED SAMPLES TEST

PAIRED SAMPLES TEST



68

CHAPTER 5 DISCUSSION AND FUTURE DIRECTIONS

Discussion

The quantitative measures of the different behaviors obtained from analyzing

video recordings of mice in Study 1 and Study 2 with the help of The Observer allowed

us to infer the following. There was no significant effect of genotype (WT, HET, PKU) on

activity and inactivity in Study 1 (p = 0.41, α=0.05) with the average time of activity

ranging from 88% to 91% and inactivity from 8 to 11% (Figure 4-1). A distinct amount of

variability was observed within the PKU data set from the box plots (Figure 4-2; Figure

4-3). This could reflect differences in Phe levels among mice in the PKU group. Mice

from both Study 1 and Study 2 spent approximately equal amounts of time being active.

These results differ from data previously published by Cabib et al.31 that report WT and

HET mice exhibiting higher levels of locomotion compared to PKU mice that are known

to experience motor disturbances. The reason for this difference in results could be a

result of differences in handling and the stress associated with a novel environment.

Rearing was defined as full rear, half rear and center rear. HET and WT mice

engaged more in full rearing compared to PKU mice (p=0.0003, α=0.05). It was

observed that WT mice did not half rear. Statistical analyses of data showed that HET

mice engaged in more half rearing than PKU mice (p=0.0001, α=0.05). It was observed

that WT and HET mice engaged in equal amounts of center rearing however PKU mice

did not exhibit any center rearing (p=0.045, α=0.05). Perhaps the somewhat smaller

size and generally poorer physical condition of the PKU mice leads to weakness of the

hind limbs and an inability to support the animal (Figure 4-4). However these results do

69

correlate with studies in the literature that report lower levels of rearing in PKU mice

when compared to the other two genotypes.31

Analysis of line cross data for Study 1 showed that WT and HET mice engaged

more in small movements and were on the whole less sedentary as compared to the

PKU mice (Figure 4-9). Post hoc pair wise comparison tests run showed that PKU mice

spent significantly lesser time engaging in smaller movements than WT and HET mice

(p=0.0001, α=0.05).

Analysis of grooming patterns in the WT and PKU mice yielded interesting results.

It was observed that they did not adhere to the synctactic grooming pattern59 and

exhibited short and incomplete bouts of paw licking and body licking. Very few mice

exhibited large bilateral strokes (Figure 4-10). We do not understand these differences

in the BTBR inbred strain from those reported for other inbred strains of mice.59 Mice in

both WT and PKU groups hardly engaged in any grooming activities and thus were not

subject to statistical analysis. Due to the small number of animals that were analyzed in

both WT and PKU groups no statistically significant differences between the two groups

could be established for the data obtained.

No statistical significance in activity or inactivity was observed among the two

treatment groups in Study 2(Activity p =0.734; Inactivity p =0.770) with the average time

of activity ranging from 67% to 77% and inactivity from 21% to 31% (Figure 4-11).

For Study 2, it could be seen from the bar graph depicting data distribution for

rearing data that mice exhibited more rearing (full rears and half rears) when they were

placed on treatment than when they were taken off treatment (Figure 4-12). No

70

statistical significance was obtained due to the small sample size (Full rear p=0.076,

α=0.05; Half rear p=0.334, α=0.05).

Statistical significance between on treatment and off treatment groups was not

obtained for line cross due to a small sample size (p=0.164, α=0.05 ) however a very

distinct decrease in small movements was observed when mice were taken off

treatment which can be seen in the bar graph depicting distribution of line cross data.

Therefore the mice seemed to be more mobile when the PAL treatment was

administered and more sedentary when taken off treatment (Figure 4-13).

Future Directions

The most important changes that need to be made is to increase sample size for

each of the different groups in order to obtain statistically significant results. Preferably

around 10 mice should be used per experimental group in order to be able to run tests

that establish statistical significance using SAS and SPSS software. However this is

logistically difficult since these are long term studies and are also extremely expensive.

These mice need to be repeatedly injected with PAL during the study, ideally daily

which would be labor intensive and also requiring substantial amounts of PAL enzyme.

Phe levels in these mice need to be consistently checked which involves repeated

bleeding of the mice. These mice are also not as healthy as WT BTBR and HET mice

and have short life spans resulting in the loss of some mice at the end of the study. All

mice need to be recorded before, during and after treatment. After enzyme treatment is

stopped it takes up to 2 months for reversal of drug effects to occur after which all mice

are once again recorded. These recordings are then all analyzed to determine

differences in behavior on and off treatment. The above factors thus influenced the

mouse group size chosen for this study.

71

For further observational studies, alterations in the experimental setup should be

made to ensure optimal conditions for video recordings. All recordings should be made

from the bottom of a fogged cylindrical plexiglass chamber with even light distribution.

This may help prevent outside disturbances from influencing the behavior of the mice.

Mice should be introduced into the observational chamber before actual recordings are

made in order to acclimatize them to the novel environment. This could help in reducing

the effects of stress that a novel environment inflicts on the mice. All recording sessions

should be of equal duration and should at least be 10 minutes long. Object recognition

tests could be carried out in order to better asses the cognitive deficits exhibited by PKU

mice.

Grooming characteristics exhibited by BTBR mice did not match the grooming

patterns observed in other mice strains. The grooming behavior exhibited by WT, HET

and PKU mice deviates strongly from the traditional grooming synctactic pattern.59 They

terminated grooming behavior prematurely (not completing the entire chain Figure 3-2)

exhibiting short but repeated bouts of paw licking interspersed with some body licking

and a few large bilateral strokes. They do no follow the exact sequence of grooming

events as depicted by the traditional grooming synctactic pattern; instead they often

jump between paw licks, body licking and unilateral strokes in a haphazard fashion. This

strongly deviates from results published by Cabib et al.31 that report PKU mice engaging

in more grooming than WT and HET mice. This aberrant behavior could be due to

stress induced from the novel environment, outside disturbances or simply an intrinsic

characteristic of the BTBR strain. Abnormalities in the corpus callosum of PKU mice

were shown in studies carried out by Jackson laboratories. This could have some effect

72

on the abnormal behavioral patterns exhibited by these strain of mice even though their

studies do not pertain directly to PKU and its treatment. Further analysis of grooming

patterns in both Study 1 and Study 2 should be carried out to further explore the

aberrant grooming traits exhibited by these mice.

73

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http://www.bmrn.com/pipeline/peg-pal-for-pku.php

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BIOGRAPHICAL SKETCH

Padmini Ashok Kumar was born in Kerala India. She graduated from S.B.O.A

Matriculation and Higher Secondary school, Chennai India. She enrolled at Anna

University where she earned a B.Tech degree in Industrial Biotechnology in 2008. She

completed her undergraduate project focusing on cervical carcinoma at Madras Institute

of Technology in collaboration with Apollo Hospitals, Chennai India. She also interned at

Dr. Reddy’s Laboratories Limited before enrolling as a master’s student at the University

of Florida to obtain a degree in medical sciences. After graduation she will go on to work

in the pharmaceutical industry before pursuing an MBA.

behavioral assesment of the pahenu2 mouse...

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