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-Appoptotic 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

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 PrPgene

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 inoculcated with brain homogenates from scrape infected animals demonstrated unusual spiking patterns in the electroencephagram (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 familiar 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 conciousness, 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 somnogens (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 fore brain 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 endoplastic 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 iassociated 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 electroenencephalogram (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 Creutzfield 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 residue 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 PQGGGTWGQ (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-lhelices unique among all naturally occuring alpha helices (Morrissey and Shakhnovich 1999). Prion proteins have electrostatic interaction and saltbridges stimulated by molecular dynamics. The electrostatic interaction in general and salt bridges in part icular 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 hamnster protins they are at position 183 and 197.

Prion protein is a cell surface protein anchored by a glycosylphosphatidylinostol anchor (GPI). This is anchored to sphingolipid Rafts (membrane Organisation of GPI-APS into a laterally organized cholesternsphingolipid ordered membrane domain). From this sphingohipid 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 dimentional 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 gobular 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 glycosylphosphatodylinositol (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 . PrPsen on the cell surface has a metabolic half-life of 3-6 hours (Borchelt et al, 1990) and most PrP appear to be degraded in non-acidic compartments bound by cholesterol-rich membrane.

Studies on endocytosis of PrPsen indicate that it cycles between the cell surface. Both sulfate glycans and copper have been shown to stimulate the endocytotic compartment with transit time of 60 minutes endocytosis process (Pauly and Harris, 1998). However, the exact mechanism of internalization has been controversial. The GPI anchors of PrPc determines its route (Taraboulos et al, 1995)

Endocytosis remains the process in which materials (in this case, protein) enter the cell without passing through the cell membrane. The membrane folds around the protein outside the cell resulting to the formation of a sack like vesicle into which the material (Protein) is incorporated in this way. A macromolecule such as protein is said to be internalized in the existing component of cells. Numerous mammalian proteins have a special post-translational modification at their carboxy-terminal known as the glycosylphosphatidylinsitol (GPI) anchor, which serves to attach the proteins to the extra cellular leaflet of the cell membrane. The GPI anchor consists of a phosphatidylinsitol group attached to a carbohydrate moiety (trimanosyl-nonactylated glucosamine), which in turn is linked through a phosphodiester bond to carboxy-terminal amino acid of the mature protein. Glycosylphosphosphatidylinsitol anchored proteins (GPI-APS) therefore, represents an interesting amalgamation of the three basic kinds of cellular macromolecules, viz, proteins, carbohydrates and lipids. For prion proteins, the cell surfaces were shown to trafic through the endocytic intermediates and this step was even shown to be necessary for conversion of PrPc to PrPsc. Clathrin coated pits were shown to be instrumental to the endocytosis of PrP. Through this process of endocytosis and internalization, molecular materials are attached to the structures of prion protein.Molecular dynamic stimulation suggest that some attached N-glycans may modulate PrPc stability (DeMarco and Daggett 2005; Ermonval et al., 2003).

Cellular prion protein (PrPc) can be liberated from the cell surface invitro by enzyme phosphoinositol phospholipase (PiPl), which usually cleave the phosphatidylinostol glycolipid anchor (Weisman, 1999). PrPc is readily digested by proteinase K, it is designated with the term PrPsen. Full-length recombinant proteins and as prion protein (amino acid residue 23-231 is denatured by neutral salts such as sulfate and flouride salts, contrary to the report that structure of protein, either basic or acidic are stabilized against denaturation by certain neutral salts such as sulfate and fluoride (Nishimura et al, 2002). Under identical concentration of neutral salts, the structure of sheep prion protein which contains a greater number of glycine groups in N-terminal unsaturated segment than mouse PrP becomes more stabilized. Also in contrast to full-length protein, the C-terminal 121-231 prion protein fragment consisting of all the structural elements of the protein i.e. three α-helices and two short β-strands is stabilized against denaturation by neutral salts. Prion protein has a preferential interaction with glycine residues in the N-terminal segment, consistent with α-helix I. The prion α-helix I is the most soluble of all the prion α-helices reported so far in literature. Increasing the concentration of anions on the prion protein surface perturbs the solubility of the α-helix I, thereby making structural conversion of protein structure to β-pleated sheet (insoluble) by anionic nucleic acid . It is equally reported that DNA can also modulate the aggregating properties of prion protein (Cordeiro et al., 2001). Interaction between prion protein and nucleic acid also leads to the demonstration that prion protein can play a role in nucleic acid metabolism (Gabus et al., 2001). PrPc has a molecular weight of 35-36 KDA and very hydrophilic alpha helical structure. It can pass through the fliter paper with an average pore diameter of 20-100nm, suggesting a size range consistent with conventional viruses (Eklund et al., 1963). It has sedimentation constant ranging from 200 S to 2000 S. (Prusiner et al., 1977). Prion protein is highly expressed within the nervous system, although its content varies among distinct brain regions. It is predorminantly expressed in the brain, spinal cord and lymphoid tissues (spleen, lymph nodes and thymus) (Chio et al., 2006). The protein can also be expressed in decreasing amount in various components of immune system, salivary glands, lungs, bone marrow, blood, intestine, liver, kidney, uterus and peripheral tissues (Eklund et al., 1967). The expression of PrPc by neurons within the central nervous system is particularly in the hippocampus, neocortex, spinal motor neuron, and cerebella, purkinje cells (Piccardo et al., 1990; Sales et al., 1998). Modest amounts of PrPc are also expressed in glial cells within the brain and spinal cord, in peripheral tissues and in human T-cells. B-cells, monocytes and dendrites cells but not as much in blood cell. Immunoblotting studies revealed that PrPc glycoforms and the composition of N-linked glycans or PrPc in human peripheral blood mononuclear cells are different from those of the brain or neuroblastoma cells (Ruliang et al., 2001).

1.3.1        Functions of Prion Protein

Nature has roles or functions vested on the cellular prion protein for their expressions on various cells, tissues and organs of the humans and other mammals. Although, some of these functions are not yet clearly elucidated, some experimental demonstrations are evident.

1.3.2        Prion Protein and Cell Membrane Viability

The widely reported and accepted theory that cellular prion protein (PrPc) is anchored to the cell surface and invariably on the neuronal surface by glycosylphosphatidylinositol, suggests a role in the cell signaling or adhersion. It is reported that the hippocampal slices from the PrP null mice have weakened GABAA (γ-amino butric acidA) receptor-mediated fast inhibition and impaired long term potentiation. This impaired synaptic inhibition may be involved in the epileptic form activity seen in Creutzfield Jacob disease. Therefore it is argued that loss of function of PrPc on the neuronal surface may contribute to the early synaptic loss and neuronal degeneration (Oesch et al., 1985; John-collinge et al., 1994).

1.3.3 Prion Protein and Sleep

In an experimental design, it was demonstrated that after sleep deprivation PrP null mice showed a larger degree of sleep fragmentation and latency to enter rapid eye movement sleep and non rapid eye movement. Also during sleep recovery experiment the amount of NREM sleep and the SWS were reduced in PrP null mice. This finding demonstrated that neuronal PrPc is involved in sleep homeostasis and sleep continuity while non-neuronal PrPc is not involved (Manuel et al., 2007).

Altered sleep pattern and circadian activity rhythm have been observed in mice devoid of PrP (Tobler et al., 1997). There are some evidences that fatal familial insomnia, an inherited human prion disease results from deficiency of normal prion protein, a deficiency that occurs because mutant prions are unable to fulfill normal functions. PrPc null mice were observed to develop normally at early stage of life but underwent severe ataxia and purkinje cell degeneration at advanced ages (Manuel et al., 2007). Impaired coordination observed in aged PrP null mice (70 weeks and above) was attributed to lack of PrPc and correlated with the loss of cerebella purkinje cells in PrP null mice. Purkinje cells survive longer with the presence of cellular prion protein (Katamine et al., 1998). They showed a slight increase in locomotor activity during exploration of environment. Also under acute stress, such as restraint or electric footshock, mice lacking PrP showed reduced levels of anxiety when compared to the PrP expressed mice. Anxiety is accompanied by a characteristics set of behavioural and physiological responses that tend to protect the individual from danger and is taken as part of a universal mechanism of adaptation to adverse condition (Chen et al., 1995).

1.3.4        Anti-Appoptotic Function

Cellular prion protein has anti-apoptotic effects. It plays a role against Bax-mediated neuronal apoptosis. Bax-mediated apoptosis refers to the Bcl-2 associated protein-X (Bax) mediation apoptosis. PrPc potently inhibits Bax-induced cell death in human neurons. Deletion of four octapectide repeat of PrPc by mutation or otherwise completely or partially eliminates the neuro protective effects of PrPc. PrPc remains anti-apoptotic despite truncation of glycosylphosphatidylinositol anchor signal peptide, indicating that neuro protective form of normal prion protein does not require the abundant cell surface GPI anchor PrP (Bounher et al., 2001; Kuwahara et al., 1999). It was also reported that neuronal PrPc engagement with stress-inducible protein-1 and laminin plays a key role in cell survival and differentiation. This was demonstrated from the PrPc expression in astorcytes (Star shaped glial cells in the brain and spinal cord).The study evaluated whether PrPc expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wilde-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrPc null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrPc null astrocytes and neurons. Laminins (LN) (Major proteins in the basal lamina, a protein network foundation for most cells and organs) hold cells and tissues together.They are secreted and deposited at the extracellular matrix by wild type astrocytes; presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, laminin coming from PrPc null astrocytes displayed a punctuate distribution, and it did not support neuronal differentiation.

Additionally, secreted soluble factors from PrPc-null astrocytes promoted lower levels of neuronal survival than those secreted by wild type astrocytes. PrPc and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrPc expression in astrocytes is critical for sustaining cell to cell interactions, the organization of extracelluar matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival (Lima et al., 2007). PrPc also interact with laminin for its function of memory processing, consolidation/retention and cognitive performance in mammals, especially humans. It was demonstrated that hippocampal PrPc plays a critical role in memory processing through interaction with Laminin. One of the plausible hypotheses is based on the interaction of laminin with tissue type plasminogen activator/plasmin proteolytic cascade. On the other hand, laminin stimulates neurite outgrowth, and the most abundant laminin isoform in the hippocampus is LN10 (α5

β1γ1), which is produced and secreted by neurons. These cells bind to LN10 through intergrin α3β, as well as through PrPc. The PrPc binding domain maps to the COOH-terminal domain of lamininγ-1 chain, and only PrPc binds to this domain,through which it is able to promote neurite outgrowth (Indyk et al., 2003). PrPc interaction with laminin is also involved in the neuronal signaling process and signal transduction in neuronal cells (Spielhaupter and Schatzl, 2001).

It has been reported that PrPc plays a key role in maintaining myelin, a fatty substance that forms a sheet around nerves and helps transmit nerve signals. It was also found that mice without PrP in certain nerve cells suffer from a demyelinating disease that closely resembles one seen in humans. A published paper which suggested that mice without PrP had damage to their peripheral nerves, triggered a scientific probe on this report. A team of scientists examined five strains of mice lacking the PrP gene and found that all showed this peripheral nerve damage by ten weeks of age. Since this finding did not actually answer what was behind the nerve damage, a one- year old mice was studied and found that their sciatic nerve (the large nerve in the back that runs into the legs) had lost myelin. Then mice that lacked PrP in some cells but not in the others were studied to see which cells were behind the demyelination. The result was a surprise. When PrP was present on the axons (the fibers that conduct electrical impulses), it prevented disease.

When it was lacking in axons but present in the so called Schwarnn cells that actually form the myelin sheet, the mice got sick. Though the Schwarnn cell are the ones affected when PrP is missing, the protein must be present in axons to prevent disease (Radovanovic et al., 2005).

1.3.5        Protein and Immune System

Despite the involvement of specific immune cell-type in the accumulation of PrPsc in peripheral lymphoid compartments at early stages of prion disease, no attention has been paid to whether PrPc is depleted in the immune cells and possible consequences of immune responses. Some data show that PrPc may play important roles in the development and maintenance of immune system, as well as in specific cellular immunological responses (Aguzzi et al., 2003). Studies also suggest that PrP plays a role in the cultivation of lymphocytes (Li et al., 2001). As noted earlier, the cellular prion protein (PrPc) is expressed widely in immune system in haematopoietic stem cells and mature lymphoid and myeloid compartment in addition to cells of the central nervous system. It is up regulated in T-cells activation and may be expressed at higher levels by specialized classes of lymphocytes. Furthermore, antibody cross-linking of surface PrP modulates and T-cell activation, leads to the rearrangements of lipids raft constituents and increased phosphoraylation of signaling proteins. These findings appear to indicate an important, but, as yet, ill-defined role of PrPc in T-cell. Although, PrP mice has be been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self renewal of haematopoietic sterm cell (Aguzzi et al., 2003; Choi et al., 2005).

1.3.6        Prion Protein and Muscular Tone

Prion protein (PrPc) has roles or functions beyond the nervous and immune systems. Expression of PrPc is increased in sporadic and hereditary inclusions, body myositis and myopathy, polydermatomysitis, and neurogenic muscles atrophy. A uniform pattern of increased PrPc expression was described in a series of muscular disorders. Interestingly, both glycoform profile and size of PrPc in normal muscle are distinct from human brain (Kovacs et al., 2004). Based on these findings, it was suggested that PrPc may have a general stress-response effect in neuromuscular disorders (Kovacs et al., 2004). This hypothesis is supported by accumulation of PrPc in muscle fibers of an experimental model of chloroquine- induced myopathy (Furukawa et al., 2004). In addition, PrPc was up regulated when myotubes differentiate from immortalized C2C1 murine myoblasts (Brown et al., 1998). PrPc content progressively increased during maturation of myocytes in primary culture of skeletal muscle, attributed to both transcriptional and post translational changes. Fast muscle fibers present a higher concentration of PrPc than slow fibers and are consistent with a role of PrPc in skeletal muscles physiology. A severe dilated cardiomyopathy has also been described in patients diagnosed as sporadic CJD, and a heart biopsy contained evidence of the presence of PrPsc. Since no other cause was found, it was suggested that the disease is derived from accumulation of PrPsc into the heart (Ashwath et al., 2005). Recently disease associated PrP was also detected in cardiac myocytes of elk and white-tailed deer infected with chronic wasting disease(CWD),but the heart physiology was not evaluated (Jawell, et al, 2006). These data raised the thought that PrPc may have important functions in both skeletal and cardiac muscles.

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