DETERMINATION OF PRION PROTEIN (PrP) AND CHANGES IN FERTILITY HORMONES WITH SLEEP DEPRIVATION IN ALBINO RATS

ABSTRACT

Wistar albino rats, numbering thirty five (35), were nurtured in the animal house of University of Nigeria Enugu Campus and used for this work. This work is designed to determine the presence of prion (PrP) in Wistar albino rats and the possible changes that sleep deprivation can cause in PrP and fertility hormones. Twenty four (24) of the animals (15 females 9 males) were successfully sleep-deprived for 14 days while 11 were not deprived of sleep. The non-sleep deprived rats were used as a control group in addition to PrPc commercial control, for the prion protein determination. The body weights of the rats were obtained before and after sleep deprivation. Serum samples were collected before and after sleep deprivation for the fertility hormone assay while brain tissues were extracted from each sleep deprived and non-sleep deprived rat after 14 days for prion protein determination and histological studies. Single platform sleep deprivation technique was used for sleep deprivation, ocular venipuncture for blood collection, euthanization for sacrificing the rats and enzyme linked immunosorbent assay method for both hormone assay and prion protein determination respectively. Part of the brain tissues were prepared histologically (sectioning and staining) using congo-red staining technique for possible sleep deprivation induced morphological changes. The presence of PrP as determined, was confirmed by comparison of the values of the two control groups and test samples while a significant increase (p < 0.05) in PrP concentration after sleep deprivation was observed when compared with non sleep deprived group of albino rats. Sex hormones such as testosterone and oestradiol, decreased significantly (p < 0.05). The concentrations of prolactin and thyroid stimulating hormone and the body weight of the rats also showed a significant decrease (p < 0.05) after sleep deprivation compared with the normal control rats. The concentrations of follicle stimulating hormone and luteinizing hormone had no significant (p > 0.05) changes after sleep deprivation when compared with control group of albino rats. There was decrease in oestradiol, testosterone, prolactin, thyroid stimulating hormones and body weight of rats while FSH, LH and brain tissues showed no significant changes. There were also no observable changes in the brain tissue morphology after sleep deprivation. In conclusion, there was PrPC induction following sleep deprivation in albino rats. It is therefore recommended that sleep deprivation should be put into consideration in infertility cases and more work should be done on Prion proteins for neuropathological cases.

TABLE OF CONTENTS

Title Page
Certification
Dedication
Acknowledgments
Abstract
Table of Contents
List of Figures
List of Plates
List of Abbreviations

CHAPTER ONE: INTRODUCTION
1.1 Sleep
1.1.1 Biology of Sleep
1.1.2 Regulation of Sleep
1.1.3 Functions of Sleep
1.2 Sleep Deprivation
1.2.1 Sleep Disorders
1.2.2 Sleep Deprivation and Associated Problems
1.2.3 Sleep Deprivation and Protein Metabolism
1.2.4 Sleep Deprivation and Prion Protein
1.3 Prion Protein (PrP)
1.3.1 Functions of Prion Protein
1.3.2 Prion Protein and Cell Membrane Viability
1.3.3 Prion Proteins and Sleep
1.3.4 Anti-Apoptotic Function
1.3.5 Protein and immune System
1.3.6 Prion Protein and Muscular Tone
1.3.7 Abnormal Prion Protein
1.3.8 The Pathogenicity of Prion
1.3.9 The Diseases of Prion (PrPres)
1.4 Endocrine System
1.5 Hormones
1.5.1 Follicle Stimulating Hormone
1.5.2 Luteinizing Hormones
1.5.3 Prolactin (PRL)
1.5.4 Thyroid Stimulating Hormone
1.5.5 Testosterone
1.5.6 Oestradiol
1.6 Body Weight
1.7 Analysis of Methodology for Sleep Deprivation, Prion Protein and Hormones Assay
1.7.1 Gentle Handling
1.7.2 Single Platform
1.7.3 Multiple Platforms
1.7.4 Modified Multiple Platforms
1.7.5 Pendulum
1.7.6 Automated sleep deprivation
1.7.7 Prion Protein Detection Methods
1.7.7.1 Western blot
1.7.7.2 Immunohistochemistry
1.7.7.3 ELISA
1.7.7.4 Staining of Amyloid Proteins
1.8 Nature of Research Subject
1.9 Consent
1.10 Aim and Objectives of the Study
1.10.1 Aim of the Study
1.10.2 Specific Objectives of the Study

CHAPTER TWO: MATERIAL AND METHODS
2.1 Materials
2.1.1 Animals
2.1.2 Chemicals and Reagents
2.1.3 Equipment
2.2 Methods
2.2.1 Sleep Deprivation
2.2.2 Blood Collection
2.2.3 Rat Sacrifice
2.2.4 EIA
2.2.4.1 EIA for Prion Protein Using Spi-Bio Kit
2.2.4.2 EIA for Follicle Stimulating Hormone (FSH)
2.2.4.3 EIA for Luteinizing Hormone (LH)
2.2.4.4 EIA for Prolactin
2.2.4.5 EIA for Thyroid Stimulating Hormone (TSH)
2.2.4.6 EIA for Testosterone
2.2.4.7 EIA for Oestradiol
2.2.5 Histological Procedure for Demonstration of Brain Tissue Morphology/Amyloid Protein
2.2.5.1 Alkaline Congo-red Method
2.3 Statistical Analysis

CHAPTER THREE: RESULTS
3.1 Prion Protein (PrP)
3.2 Follicle Stimulating Hormone Concentration of Control and Sleep Deprived Rats
3.3 Luteinizing Hormone (LH) Concentration of Control and Sleep Deprived Rats
3.4 Oestradiol (E2) Concentration of Control and Sleep Deprived Rats
3.5 Testosterone Concentration of Control and Sleep Deprived Rats
3.6 Prolactin Concentration of Control and Sleep Deprived Rats
3.7 Thyriod Stimulatine Hormone (TSH) Concentration of Control and Sleep Deprived Rats
3.8 Body Weight of Control and Sleep Deprived Rats
3.9 Brain Tissue Morphology of Control and Sleep Deprived Rats

CHAPTER FOUR: DISCUSSION
4.1 Discussion
4.2 Conclusion
4.3 Suggestions for Further Studies
REFERENCES
APPENDICES

LIST OF ABBREVIATIONS

AchE Acetylcholinesterase Enzyme

AIDS Acquired Immune Deficiency Syndrome

AMP Adenosine Monophosphate

BDI Benzodizpine

BIP Immunoglobulin Binding Protein.

BSE Bovine Spongyform Encephalopathy

CAH Congenital Adrenal Hyperplasia

cAMP Cyclic Adenosine Monophosphate

CCK Cholecystokinin

CCIP Corticotropin Intermediate Lobe Peptide.

CJD Creutzfeldt Jacobs Disease

CNS Central Nervous System

CSF Cerebrospinal Fluid.

CWD Chronic Wasting Disease

DHT Dihydrotestosterone

DNA Deoxyribonucleic Acid

DSIP Delta Sleep Inducing Peptide

E2 Estradiol

EEG Electroencephagram

EIA Enzyme Immuno Assay

ELISA Enzyme Linked Immunosorbent Assay

ER Endoplasmic Recticulum

FSH Follicle Stimulating Hormone.

fCJD Familial Creutzfeldt Disease

GC Glucocorticoid

GH Growth Hormone

GHRH Growth Hormone Releasing Hormone.

GPI Glycosyl phosphatidyl Inositol.

GRF Growth Releasing Factor

HCG Human Corionic Gondotrophin

HIV Human Immune Deficiency Syndrome

HP.A Hypothalamic Pituitary Axis

IFN Interferone

IGF Insulin Like Growth Factor

LH Luteinizing Hormone

MP Muramy Peptide

mRNA Messenger RNA

MSH Melanocyte Stimulating Hormone

NADPH Nicotinamide Adeninedinuclutide

NREM Non Rapid Eye Movement

OT Oxytocin

PGD2 Prostagladin D2

PL/PRL Prolactin.

PK Proteinase-K

PIH Prolactin Inhibiting Hormone.

PRNP/PrnP Human PrP gene

PrP Prion Protein.

PrPc Cellular Protein/Normal PrP

PrPsen Proteinase-K Sensitive PrP/Normal PrP

PrPsc Screpie PrP/Abnormal PrP

PrPres Proteinase-K Resistant PrP/Abnormal PrP

TSE Transmissible Spongyform Encephalopathy

PS Paradoxical Sleep

PSD Paradoxical Sleep Deprivation

PTH Parathyroid Hormone

PVN Paraventicular Nucleus

RAS Reticular Activity System

REM Rapid Eye Movement.

SDS Sodium Deodescyl Sulphate

SPS Sleep Promoting Substances

SWS Slow Wave Sleep

T3 Triiodothyronine

T4 Total Thyroxine

TMB Tetramethylbenzidine

TME Transmissible Mink Encephalopathy.

TNF Tumor Necrosis Factor

TPO Thyroid Peroxidase

TSH Thyroid Stimulating Hormone

TSHR TSH Receptor

VIP Vasoactive Intestinal Peptide

CHAPTER ONE

INTRODUCTION

Sleep is the natural state of bodily rest observed in mammals, birds, many reptiles, amphibians and fishes. It is equally a state of unconsciousness from which a person or animals can be aroused. In this state the brain is relatively more responsive to internal than external stimuli. In contrast, coma is also a state of unconsciousness from which a person or animals cannot be aroused (Max, 2006). Sleep is homeostatic; therefore it is controlled by the body’s internal balance (Max 2006). It is considered critical for maintenance of health, support of life, restoration of body and brain functions and promotion of neural-immune interaction (Aurell and Elqvist, 1985; Everson et al., 1989). These are reflected in the roles of sleep in the brain for memory co-ordination and teaching (Turner et al., 2007). Through its role in hormone activities such as in growth hormone, thyroid stimulating hormone and prolactin to mention a few, metabolic processes are properly co-coordinated and carbohydrate storages are carried out (Bonnet and Arand, 2003; Everson and Read, 1995).

Sleep deprivation, a general lack of necessary amount of sleep, which may occur as a result of sleep disorder or deliberate inducement or torture, is deleterious to health when it is prolonged. It has been scientifically observed that prolonged sleep deprivation may result in aching muscles, blurred vision, and clinical depression, and constipation, dark circles under the eyes, daytime drowsiness, and decrease mental activity and concentration, delirium, dizziness, fainting, hallucination, hand tremor, headache, hypertension, irritability, loss of appetite, memory lapses or loss, nausea, nystagmus, pallor, psychosis-like symptoms, severely yawning, sleep paralysis while awake, slowed reaction time, slowed wound healing, synaesthesia, temper tantrum in children, weakened immune system, weight loss, diabetes mellitus type II, obesity without weight gain and death (Gotlieb et al., 2005).

Prion protein pathologies are also associated with alteration in sleep. Rats inoculated with brain homogenates from scrape infected animals demonstrated unusual spiking patterns in the electroencephalogram (E.E.G) about four months after inoculation. During that period slow wave sleep (SWS) and active wakefulness are reduced while drowsiness is increased (Bassant et al., 1984; Bassant et al., 1986). In human, the condition known as fatal familial insomnia is associated with prion disease related to thalamic neurodegeneration (Gibbs et al., 1980). Mutation in prion protein, a glycoprotein on neuronal membrane astrocytes, may underlie the pathological changes that accompany this condition (Monanri et al., 1994). Mice that genetically lack the prion protein gene demonstrated alterations in both sleep and circadian rhythms (Tobler et al., 1997). It has been demonstrated that neuronal cellular prion protein (PrPc) (but not non-neuronal) is involved in sleep homeostasis and sleep continuity (Manuel et al., 2007).

The main systemic disorder resulting from prolonged sleep deprivation in laboratory animals are negative energy balance, low thyroid hormones, and host defense impairment (Bergmann et al., 1989; Everson and Nowak, 2002). Prolactin, a lactating hormone and one of the anabolic hormones involved in sleep promoting activities was observed to be reduced during prolonged sleep deprivation (Vontruer et al., 1996).

Recent finding on the alterations in thyroid hormones in sleep deprived rats point to the brain as the essential site of sleep deprivation effects (Utiger, 1987). The hypothalamus and pituitary are the main sites of hormone production and regulation in the brain. Relatively, little is known regarding other neuro-endocrine consequences of sustained sleep deprivation and whether there is broad pituitary or hypothalamic involvement. It has also become necessary to survey the possibility of changes in the levels of some fertility hormones with sleep deprivation. The hormones of interest here are the follicle-stimulating hormone (FSH), luteinizing hormone (LH), ooestradiol, testosterone, Prolactin and thyroid-stimulating hormone (TSH).

Following the various relationships between sleep deprivation, prion protein (PrP) and hormones, it is necessary to explore the possible changes sleep deprivation may induce on PrP and some fertility hormones.

1.1 Sleep

In animals, sleep is a naturally recurring state characterized by altered consciousness, relatively inhibited sensory activity, and inhibition of nearly all voluntary muscles. It is distinguished from wakefulness by a decreased ability to react to stimuli and it is more easily reversible than being in hibernation or a coma (Macmillian, 1981). Sleep is the natural state of bodily rest observed in mammals including humans. It is also observed in birds, many reptiles, amphibians and fishes. It is common to all mammals and birds. It is equally a state of unconsciousness from which a person or animal can be aroused. In this state, the brain is relatively more responsive to internal than external stimuli. The unconscious state of sleep is distinguished from that of coma by the fact that unconsciousness of coma in mammal or animal cannot be aroused (Max 2006; Ursin, 1983).

1.1.1 Biology of Sleep

Sleep was thought to be a passive state but it is now known to be a dynamic process. It is homeostatic, therefore, it is controlled by the body’s internal balance.The brain is the seat of internal balance and it is active even during sleep. The brain is made up of parts and nerve centres that elicit nerve-signaling chemicals called neurotransmitters. The state of the brain activities during sleep and wakefulness results from activating and inhibiting forces that are generated within the brain. The neurotransmitters such as serotonin and norepinephrine of the brain control whether one sleeps or keeps awake by acting on the nerve cells or neurons in different parts of the brain as the need arises. The frontal lobe of the brain keeps the body awake. It is the centre of planning, the memory search, motor control and reasoning. The thalamus is for attention and sleep. The hypothalamus, located under the thalamus plays the role of promoting the type of sleep called slow wave sleep (SWS). The brain stem plays a great role in sleep and wakefulness. The brain stem is a set of neural structures at the base of the brain. It connects the brain to the spinal cord. It is made up of the medullar, the pons and the reticular formation. While the reticular formation helps to keep, the body awake and alert, the pons is involved in the sleep and control of facial muscles. The neurons at the brain stem actively cause sleep by inhibiting other parts of the brain that keep a person or animal awake (Sherwood, 1997).

1.1.2 Regulation of Sleep

Sleep, one of the most sophisticated integrative functions in higher animals, appears to be regulated by the brain in conjunction with a variety of endogenous humoral factors. These factors are called sleep substances (Inoue, 1985). These substances are endogenous in the brain, cerebrospinal fluid and blood. These substances under the high physiological demand for sleep in the organism are produced in the brain stem and transferred to the whole brain via the body fluid (especially CSF) to induce or maintain sleep. These substances include peptides or protein, hormones and somnogenic (Pappenleiner, 1975; Schoeneberger, 1977).

It is well known that growth hormones (GH) is secreted during delta sleep at first few periods of sleep cycle in humans (Gronfier et al., 1996). It equally plays a part in subsequent appearances of rapid eye movement (REM). Prostaglandin D2 (PGD2) has been revealed as one of the most promising candidates for an endogenous sleep substance. It induces slow wave sleep (SWS) in rats under restrained conditions (Obal, 2003). Adenosine, a purine nucleoside produced during nucleic acid metabolism and protein catabolism builds up in our blood when we are awake. At a level of accumulation, it stimulates drowsiness/sleep and break down gradually when we are asleep to enable restoration of wakefulness (Obal, 2003). Other substances such as emphetamines, caffeine, cocaine and crack cocaine, energy drinks and methylphenidate cause wakefulness (Abaraca et al., 2002). Wakefulness actually refers to a period of consciousness. The term consciousness therefore refers to subjective awareness of private inner world of one’s own mind, that is, awareness of thoughts, dreams and events. Maximum alertness depends on attention and getting sensory impute that energizes the reticular activity system (RAS) of the reticular formation of the brain stem and subsequently the activity level of the central nervous system (CNS) as a whole.

1.1.3 Functions of Sleep

Sleep is considered critical for maintenance of health, support of life, restoration of body and brain functions and production of neural interaction (Aurell and Elqvist, 1985; Everson et al., 1989). These are reflected in the roles of sleep in the brain for memory consolidation and learning. Working memory is important. It keeps information active for further processing and support higher-level cognitive functions such as decision making, reasoning and episodic memory. These functions were shown to be affected by sleep deprivation in humans to a drop of about 38% in comparison to non-sleep deprived individuals (Turners et al., 2007).

Through its roles in hormonal activities, such as growth hormones, thyroid stimulating hormones and prolactin to mention but a few, metabolic processes are properly coordinated and carbohydrate storages are carried out (Bonnet and Arand, 2003; Bergmann et al, 1989 and Everson and Read, 1995). It has been shown that sleep, more specifically slow wave sleep, does affect growth hormone levels in adult men. During eight hours sleep it was found that the men with high percentage of SWS (average 24%) also had low growth hormone secretion while subjects with a low percentage SWS (average 9 %) had high growth hormone secretion. There are multiple arguments supporting the restorative functions of sleep. We are rested after sleeping and it is natural to assume that this is a basic purpose of sleep. The metabolic phase during sleep is anabolic and anabolic hormone such as growth hormones as mentioned earlier are secreted preferentially during sleep (van Cauter et al., 2000).

1.2 Sleep Deprivation

Sleep deprivation is a general lack of the necessary amount of sleep. This may occur as a result of sleep disorders, active choice or deliberate inducement such as interrogation, for purposes of keeping watch for security reasons, prolonged study or research and some times for torture. During sleep deprivation there is a progressive increase in peripheral energy expenditure to nearly double normal levels, resulting to negative energy balance (Everson and Wahr, 1993). In response to this metabolic demand, an increase in serotonin and catecholamines act on both the frontal lobe of the brain stem to keep the body awake (NIH Pub, May 2007).

1.2.1 Sleep Disorders

The actual sleep disorders include sleep apnea (apnoea), narcolepsy, primary insomnia, periodic limb movement disorder, restless leg syndrome and the circadian rhythm sleep disorders. Sleep apnoea is caused by lack of Co2 tension in the blood for stimulation of the respiratory centre which in turn causes failure of the autonomic control of the respiration. This becomes more pronounced during sleep. Narcolepsy is the sudden, repetitive attack of sleep occurring in the daytime, causing diverse clinical conditions. Body pains, illness, stress and drugs can equally cause sleep deprivation during such conditions. Elderly people may loose ability to consolidate sleep due to aging factors.

1.2.2 Sleep Deprivation and Associated Problems

When the body is deprived of sleep for a long time, it elicits a number of negative responses resulting to a number of diseases (Rechtcheffen, 1983). Such responses include negative energy balance, protein malnutrition reduction in circulating anabolic hormones and host defense impairment (Everson, 2004). Though food consumption remained normal in sleep deprived rats fed with a diet of high protein-to-calorie ratio, body weight loss was more than 16% of baseline, development of skin lesions was hastened and longevity was shortened 40% compared with sleep deprived rats fed with the calorie augmented diet. Food consumption of the calorie fed rats was lower during baseline than that of protein fed group but during sleep deprivation increased to amounts 250% of normal without net body weight gain, implying negative energy balance and malnutrition during prolonged sleep deprivation (Everson and Wehr, 1993). The negative energy balance is not due to malabsorption of calorie or diabetes but may be a metabolic response to infectious processes (Everson and Crawley 2004). In a study by Zager and co-workers, rats deprived of sleep for 24 hours were found to have 20% decreases in white blood cell count when compared with the control group (Zager et al., 2007). It was equally shown that in prolonged sleep deprivation or sleep loss, there is a progressive increase in circulating phagocytic cells, mainly neutrophils, migrating into extra vascular liver and lung tissues. These are consitent with tissue injury or infection and are of significant changes in immune system. Also, it was noted that sleep deprivation and strainous exercise result to decrease in neutrophils, monocytes, Eosinophils and lymphocytes. Also major subgroups of immune factors such as CD4, T cells, CD8 T cells, B cells and NK cells were reduced. Furthermore, Cytokines, low molecular weight proteins whose receptors are produced in Central Nervous System (CNS) which mediate many aspects of the host defense, inflammation and tissue remolding and also powerful modulators of sleep-wake behavior are altered during sleep loss in response to microbial infections (Opp and Toth, 2003). The hypothesis that chronic sleep loss impairs immune competence is most strongly supported by observation that chronic sleep deprivation in rats results to intestinal bacterial proliferation, microbial penetration into the lymph nodes, septicemia and eventual death (Opp and Toth, 2003). Conversely, experimental challenges tests have shown that bacterial products and particular immuno modulators such as Cytokines and Chemokines can alter the amount of sleep and its stages (Krueger et al., 2001; Obal and Krueger, 2003). The demonstrated link between cytokines and sleep was the observation that sleep deprivation enhanced the ability of leucocytes antiviral interferon (IFN), which has a role in modulation of sleep. The type I interferon are well known as antiviral cytokines and may be particularly important as modulators of viral induced alterations in sleep. However, both type1 (alpha/bets IFN) and type II (Gamma IFN or immunocytes) are known to modulate sleep. Also influenza, immune deficiency viruses (HIV, in human, FIV in cat and NDV in mice), all induce sleep alteration (Opp and Toth, 2003; Norman et al., 1990).

1.2.3 Sleep Deprivation and Protein Metabolism

Protein malnutrition and malformation are part of the negative effects elicited by prolonged sleep deprivation. Sleep is associated with increased protein synthesis in several brain regions as well as the whole cerebrum (Ramm and Smith 1990). Sleep deprivation on the contrary reduces the level of certain proteins in the rats basal forebrain and hippocampus (Basheer et al., 2005). As stated earlier, sleep deprivation affects various aspects of protein including metabolism and translational changes involving unfolding and misfolding of proteins (Schroder and Kaufmann, 2005; CiIrelli et al.,2006).

However, sleep deprivation promotes endoplasmic reticulum stress hormones and production of eif2 and membrane proteins (Proud, 2005). All components of unfolded protein response (UPR) or endoplasmic response (ER) stress were found after 6 hours of sleep deprivation in mouse neocortex, including increase in P-eif2α as well as free BIP, GRP78 and phosphrylated protein kinase-like ER kinase (PERK), a key kinase that phosphorylates eiF2α (Naidoo et al., 2005). During prolonged sleep deprivation, further changes such as transcript coding for several immunoglobulins, stress response protein such as macrophage inhibitor factor-related protein 14, heat-shock protein 27, alpha-β-crystallin and minoxidil sulfotransferase, globins and cortistatin are observed. At molecular level also several plasticity-related genes were strongly induced after acute sleep deprivation only and several glial genes were down regulated in both acute and long-term sleep deprivation conditions but to different extents. These findings suggest that sustained sleep loss may trigger a generalized inflammatory and stress response in the brain (Cirelli et al., 2006). It has equally been identified that endoplasmic reticulum(ER) resident chaperon, immunoglobulin binding protein (BIP) increase with sleep deprivation. The endoplasmic reticulum is the key cellular marker and master regulator of signaling path way called ER stress response or unfolded protein response (Naidoo et al., 2005).

1.2.4 Sleep Deprivation and Prion Protein

Prion protein related pathologies, which are associated with protein misfolding and neurodegenerative disease of the brain, are also associated with alteration in sleep (Gibbs et al., 1980; Monari et al., 1994). Rats inoculated with brain homogenates from scrapie-infected animals demonstrated unusual spiking patterns in the electroencephalogram(EEG) about four months after inoculation. During those periods, slow wave sleep (SWS) and active wakefulness are reduced while drowsiness is increased (Bassant et al., 1984; Bassant et al., 1986). Cats inoculated intracerebrally with brain homogenates from a human infected with Creutzfeldt Jacobs disease, demonstrated increased SWS time, reduce wakefulness and abnormal EEG after 20 minutes of inoculation. In human, the condition known as fatal familial insomnia is associated with prion disease related to thalamic neurodegeneration (Gibbs et al., 1980; Monari et al., 1994). Mutation in prion protein, a glycoprotein on neuronal membrane astrocytes, may underlie the pathological changes that accompany this condition (Monari et al., 1994). Mice that genetically lack the prion protein gene, demonstrate alterations in both sleep and circadian rhythms (Tobler et al, 1997). PrP-null mice have a low sleep pressure, leading to more frequent interruptions of sleep and reduced SWS (Tobler et al., 1997). In other words, the PrP-null mice (PrP %) show longer sleep fragmentation together with an increase of slow wave activity (SWA) during NREM sleep after a short period of sleep deprivation. It has been demonstrated that neuronal cellular prion protein (PrPc) but not the non-neuronal, is involved in sleep homeostasis and sleep continuity (Manuel et al., 2007; Tobler et al., 1997).

1.3 Prion Protein (PrP)

Prion protein is a special type of protein that is present in al mammals. It is encoded by a sinc gene at chromosome 20 (Dickson et al., 1968). Prion protein is expressed predominantly in the brain, spinal cord and lymphoid tissues (spleen, lymp nodes and thymus) (Chiol et al., 2006). The protein can also be found in decreasing amounts in salivary glands, lungs, intestines, liver, kidneys uterus and blood (Eklund et al., 1967). Prion protein is a cell surface protein, anchored by a glycosylphosphatidylinostol anchor (GPI) (Oesch et al., 1985). Prion protein can be found in its natural or normal state referred to as cellular prion protein and designated as PrPc. This cellular prion protein (PrPc) is readily digested by proteinase K, just like other common proteins. Owing to its sensitivity to proteinase k. it is also designated PrPsen. The cellular prion protein (PrPc) can be transformed to abnormal form called prions. Prions are resistant to proteinase k digestion and are therefore designated as PrPres. These prions (PrPres) are the only proteinacious particles that cause disease in vertebrates (Chesebro, 1990).

Prion protein has three-dimensional structure like other proteins. It has highly positively charged N-terminal segment. The N-terminal segment comprising residues 23 – 125 of the protein is flexibly disordered. The N-terminal segment contains four octapeptide repeats, PHGG (G/S) WGQ (between residues 60-93) and a homologous sequence lacking a histidine residue PQG G WGQ (Between residue 52 and 60). It equally has globular fragment. The globular C. terminal fragment 121-231 contains three α- helices and two β-strands (Riek et al., 1996). The hydrophilicity and charge distribution make the first prion protein α-helices unique among all naturally occurring alpha helices (Morrissey and Shakhnovich 1999). Prion proteins have electrostatic interaction and salt bridges stimulated by molecular dynamics. The electrostatic interaction in general and salt bridges in particular plays an important role in prion protein stability. This protein is coded in all mammals by a sinc gene (Dickson et al., 1968). In man, it is transcribed by a sinc gene present on chromosome 20. The molecular structure of prion protein is dictated by the prion gene, the human form of which is abbreviated as PrnP. PrnP encodes for a protein of 254 amino acids in length. PrnP undergoes post-translational modification in two important ways, cleavage and glycosylation. The glycosylation is at two sites. In hamster proteins they are at position 183 and 197.

Prion protein is a cell surface protein anchored by a glycosylphosphatidylinositol anchor (GPI). This is anchored to sphingolipid Rafts (membrane Organisation of GPI-APS into a laterally organized cholesterol sphingolipid ordered membrane domain). From this sphingolipid raft it can be endocytosed by a copper 2 ion activated mechanism (Oesch et al., 1985; Brown, 2001). Following the cleavage of a 22 amino acid signal peptide, mammalian cellular prion protein (PrPc) is exported to the cell surface as N-glycosylated Glycosylphosphatidylinositol (GPI) anchored protein of 208-209 amino acids, with its three dimensional structure retained (Calzolai et al., 2005; Riek et al., 1996; Zahn et al., 2000). PrPc contains an NH2-terminal flexible and random coil sequence of 100 amino acids and COOH- terminal globular domain of about 100 amino acids. The globular domain of the human PrPc is arranged in three α helices corresponding to amino acid 144-154, 173- 194 and 200-228, interspaced with an antiparallel β-pleated sheet formed β- strands at residue 128-131 and 161-164. A single disulfide bond is found between Cysteine residues 179 and 214. The NH2-terminal flexible tail comprises approximately residues 23-124, and a short flexible COOH- terminal domain corresponding to residue 229-230. The DNAs of both hamster and mouse PrP encodes for polypeptides of 254 amino acids (Locht et al., 1986). However, an N-terminal signal peptide of 22 amino acids is removed from these molecules during biosynthesis (Hope et al., 1986; Bolton et al., 1987) and an additional 23 amino acids are removed from the C-terminal of the proteins during glycosylphosphatidylinositol (GPI) addition at Ser 231 (Stahl et al., 1990a), resulting to a mature PrP polypeptide of 210 residues. A single disulfide bond in PrP forms a loop (Turk et al., 1988), which contains two consensus sites for Asn-linked glycosylation at residues 181 and 197. Addition of glycans at these sites generate three main glycosylated and fully glycosylated PrP. High mannose glycans added to the protein in the endoplasmic reticulum are converted to complex or hybrid glycans in the golgi apparatus . PriPsen on th