BEHAVIORAL ASSESMENT OF THE Pah enu2 MOUSE MODEL OF PHENYLKETONURIA PADMINI ASHOK KUMAR

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1 BEHAVIORAL ASSESMENT OF THE Pah enu2 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 1

2 Padmini Ashok Kumar 2

3 To both my parents for giving me the best education I could ask for 3

4 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. 4

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 7 LIST OF FIGURES... 8 LIST OF ABBREVIATIONS... 9 ABSTRACT CHAPTER 1 INTRODUCTION Phenylketonuria History Clinical Features Enzymatic phenotype of PKU patients Biochemical phenotype of PKU patients Phenotype of treated PKU patients Maternal PKU Syndrome Phenylalanine Metabolic Pathway THE PAH ENU2 MOUSE MODEL & PAL TREATMENT Animal Model Of PKU Alternative Therapies Previous Work Done PAL Treatment Behavioral Profile of Pah enu2 Mice EXPERIMENTAL APPROACH Specific Aims Methods and Materials Subjects Behavioral Testing Experimental setup Spatial novelty test Previous Recordings The Observer Activity

6 Inactivity Rearing Line Cross Grooming Statistical Analysis RESULTS FROM THE SPATIAL NOVELTY TEST Study 1- Genotype Comparisons Study 2- Treatment With Pal DISCUSSION AND FUTURE DIRECTIONS Discussion Future Directions LIST OF REFERENCES BIOGRAPHICAL SKETCH

7 LIST OF TABLES Table page 4-1 Behavioral Analysis-STUDY Tukey s studentized range test results for full rear- p=0.0003, F= Tukey s studentized range test results for half rear-p=0.0001, F= Tukey s studentized range test results for center rear- p=0.0450, F= Tukey s studentized range test results for line cross- p<0.0001; F= Behavioral Analysis- STUDY 2 (PKU mice) Activity Data Analysis- STUDY 2 p= Inactivity Data Analysis- STUDY 2 p= Full Rear Data Analysis- STUDY 2 p= Half Rear Data Analysis- STUDY 2 p= Line Cross Data Analysis- STUDY 2 p=

8 LIST OF FIGURES Figure page 1-1 Conversion of Phenylalanine to Tyrosine by PAH Comparison of the enzymatic pathway of PAH and PAL Observational chamber Synctactic grooming pattern exhibited by mice Box plot description Distribution of activity and inactivity data for STUDY 1(WT, HET and PKU) in terms of total percentage of time Box plot depicting distribution of activity data for STUDY Box plot depicting distribution of inactivity data for STUDY 1 (p=0.4115) Distribution of rearing data for STUDY 1 (WT, HET and PKU) in terms of rate per minute Box plot depicting distribution of full rear data for STUDY 1 (p=0.4115) Box plot depicting distribution of half rear data for STUDY 1(p=0.0001) Box plot depicting distribution of center rear data for STUDY 1 (p=0.0450) Distribution of line cross data for STUDY 1 (WT, HET and PKU) in terms of rate per minute Box plot depicting distribution of line cross data for STUDY 1 (p<.0001) Distribution of grooming data for STUDY Distribution of activity and inactivity data for STUDY 2 (on and off treatment) in terms of percentage of time Distribution of rearing data for STUDY 2 (on and off treatment) in terms of rate per minute Distribution of line cross data for STUDY 2 (on and off treatment) in terms of rate per minute

9 LIST OF ABBREVIATIONS 6-PTS AMPA BBB BH 4 BH 4 camp CHD DHPR GTP-CH HET HPA HPA IEM IQR LNAA MPKU NMDA PAH PAL Phe PKU PKU WT 6- pyruvoyl tetrahydropterin (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Blood Brain Barrier Tetrahydrobiopterin Tetrahydrobiopterin cyclic Adenosine Monophosphate Congenital Heart Disease Dihydropteridine reductase Guanosine triphosphate cyclohydrolase Heterozygous for the Pah enu2 mutation at the PAH locus Hyperphenylalaninemia Hyperphenylalaninemia Inborn Errors of Metabolism Interquartile Range Large Neutral Amino Acids Maternal Phenylketonuria N-methyl-D-aspartate Phenylalanine Hydroxylase Phenylalanine ammonia lyase Phenylalanine Homozygous for the Pah enu2 mutation at the PAH locus i.e. exhibits symptoms of phenylketonuria. Phenylketonuria Wild Type 9

10 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 Pah enu2 MOUSE MODEL OF PHENYLKETONURIA Chair: Philip J. Laipis Major: Medical Sciences By Padmini Ashok Kumar May 2011 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 Pah enu2 mice. 10

11 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 Pah enu2 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. 11

12 CHAPTER 1 INTRODUCTION Phenylketonuria Phenylketonuria (PKU) is one of the most commonly inherited genetic disorders, affecting approximately 1 child in every 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 (BH 4 ) 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. 12

13 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 13

14 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, 14

15 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 BH 4 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 8, 10 seen in PKU patients. 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, 15

16 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 8, 13 thereby causing decreased synthesis of sulfatides and an unstable myelin structure. 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 16

17 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 17

18 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 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 18

19 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. BH 4 is an essential cofactor required for the hydroxylation of phenylalanine to tyrosine by PAH. After the hydroxylation reaction BH 4 is converted to pterin-4a-carbinolamine. Dihydropteridine reductase (DHPR) regenerates BH 4 from quinone-dihydrobiopterin (q BH 2 ), produced from the recycling of pterin-4a-cabinolamine, and NADH ; a de novo supply of BH 4 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 (BH 4 ). 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. BH 4 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 BH 4 ; 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. 19

20 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. 20

21 CHAPTER 2 THE PAH ENU2 MOUSE MODEL & PAL TREATMENT Animal Model Of PKU The Pah enu2 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 Pah enu1 mouse model created by McDonald and colleagues 61 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 Pah enu1 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 Pah enu2 and Pah enu3 mouse models of which the former showed greater resemblance to the biochemical characteristics of 27, 28 human PKU. The Pah enu2 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 Pah enu2 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 21

22 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. Pah enu2 mice also serve as good models for maternal PKU syndrome. The female Pah enu2 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 Pah enu2 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 Pah enu2 mice as a result of restricted Phe intake. 28 Analyses of PKU mice treated with gene therapy carried out by Embury 25, 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. 22

23 BH 4 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; BH 4 supplementation helps prevent misfolding and inactivation of mutant missense proteins. Various trials have shown normal development and controlled Phe levels in patients treated with BH 4 supplementation. However, further studies are yet to be performed to assess safety of this treatment for maternal PKU. BH 4 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 Pah enu2 mouse model have shown that this form of the enzyme has increased 23, 24 blood circulating time. 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 Pah enu2 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 20, 25 complete reversal of the outcomes of the treatment were observed in two weeks. 23

24 Previous Work Done Work done previously in the Laipis lab focused on a histological assessment of the Pah enu2 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 Embury 26 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 Pah enu2 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 24

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. BH 4 responsiveness has been shown to lower Phe levels by around 60% in patients who exhibit mild hyperphenylalaninemia. 38 This treatment option is not effective 25

26 in patients who have a more severe form of classical PKU. This group of nonresponsive 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 BH 4 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 urine 41 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 26

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 47 making 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 Pah enu2 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 administration 53, 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 27

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 Pah enu2 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 Pah enu2 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 Pah enu2 and control mice (BTBR wild type or heterozygous mice). The first task involved retrieval of a food reward placed in baited 28

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 rereversal tasks were also performed. 29 Results from these tests showed no significant difference between wild type and Pah enu2 mice on the first reversal task however the Pah enu2 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 Pah enu2 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 Pah enu2 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 Pah enu2 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 Pah enu2 mice showed significant deficits in areas of simple discrimination, latent learning, habit learning, and short term memory

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) Pah enu2 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 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 al 32 involved odor discrimination and reversal studies to observe cognitive impairments in the Pah enu2 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 Pah enu2 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 Pah enu2 mice took significantly longer to finish all reversal tests in comparison to WT and 31

32 HET groups. The overall percentage of Pah enu2 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. Pah enu2 mice failed to exhibit latent learning, taking significantly longer to find water on the second day after pre-exposure as compared to the naïve Pah enu2 mice. The WT and HET mice performed significantly better than the Pah enu2 mouse. Overall conclusions obtained from these studies showed that the Pah enu2 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. 32

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 literature 31. We also expected to see the PKU mice engage in excessive grooming when compared to the other two genotypes 31. 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. 33

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 Pah enu2 mice and 3. homozygous Pah enu2 (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 predefined characteristics that are explained in detail further on in this chapter. 34

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 35

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). 36

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. 37

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. 38

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

40 OUTLIER UPPER INNER FENCE WHISKER 75 TH PERCENTILE MEAN MEDIAN 25 TH PERCENTILE WHISKER 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 75 th percentile (upper quartile) and 25 th 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. 40