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Home , Cytoskeleton, Internexin, Microtubule, Neurofilament, Tau protein, Vimentin

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 930 ISSN 0346-6612 ISBN 91-7305-X

Neurofilament Light as a Marker for Neurodegenerative Diseases

Niklas Norgren

Department of Clinical Microbiology, Immunology Umeå University, Sweden Umeå 2004

The articles published in the thesis have been reprinted with permission from the publishers

Copyright © 2004 by Niklas Norgren New Series No. 930 ISSN 0346-6612 ISBN 91-7305-769-X

Printed in Sweden by Solfjädern Offset AB, Umeå 2004

TABLE OF CONTENTS ABSTRACT 5 ABBREVIATIONS 6 INTRODUCTION 7 Neurodegenerative diseases 7 Immunochemical considerations on brain-derived markers in CSF 10 Intermediate filaments 12 13 Glial fibrillary acidic 15 Non markers 15 S-100 15 Tau 16 β- 16 14-3-3 17 basic protein 17 specific enolase 17 Consequences of degenerative or traumatic brain disorders 18 models of neurodegenerative diseases 19

AIMS OF THE THESIS 21

METHODS 22

Generation of monoclonal 22 linked immunosorbent assay 22

RESULTS 24

Paper I: Norgren N, Karlsson JE, Rosengren L, Stigbrand T. Monoclonal 24 antibodies selective for low molecular weight neurofilaments. Hybrid Hybridomics. 2002 Feb;21(1):53-9 Paper II: Norgren N, Rosengren L, Stigbrand T. Elevated 24 levels in neurological diseases. Brain Res. 2003 Oct 10;987(1):25-31. Paper III: Norgren N, Stigbrand T. Cerebrospinal fluid levels of 25 neurofilament light in chronic experimental autoimmune encephalomyelitis. Submitted to Brain research Bulletin. Paper IV: Norgren N, Sundström P, Svenningsson A, Rosengren L, 26 Stigbrand T, Gunnarsson M. Neurofilament and glial fibrillary acidic protein in . Neurology, 2004. 63(9): p. 1586-1590.

DISCUSSION 26

Production and characterization of anti NF-L antibodies 27 Establishment of a highly discriminative NF-L ELISA 28 Neurofilament measurements in an animal model of MS 29 MS and prognostic impact of brain-marker levels in CSF 30 Brain proteins as biomarkers of neurological diseases 32

CONCLUDING REMARKS 39

ACKNOWLEDGEMENTS 40

REFERENCES 42

PAPERS I-IV 71

ABSTRACT

Neurofilaments are the main cytoskeletal constituents in neuronal cells. They are belived to be important for maintaining the structural integrity and calibre of and thereby influencing the conduction velocity of nerve impulses.The neurofilament chains are divided into three groups according to their molecular size, neurofilament light (NF-L), neurofilament medium (NF-M) and neurofilament heavy (NF-H). The neurofilaments are obligate heteropolymers in vivo in which NF-L forms the backbone to which the heavier chains copolymerize to form the 10 nm neurofilament fibre. Different degenerative processes in the brain raise significant interest owing to the increasing mean age in the western world. Such diseases include amyotrophic lateral sclerosis, vascular , frontal lobe dementia, progressive supra-nuclear paralysis, , low pressure hydrocephalus, and multiple sclerosis (MS). We have been able to generate six highly specific monoclonal antibodies for NF-L, and four independent were elucidated using Biacore and V8 degradation. 2:1 and 47:3 were selected components in a two- site ELISA assay for detection of NF-L in body fluids owing to their outstanding abililty to bind the antigen. The assay has a least detectable dose of 60 ng/l and a standard range of 60 to 64 000 ng/l. The assay was validated on its ability to detect changes of NF-L levels in CSF in patients with different neurological diseases. These were cerebral infarction, amyotrophic lateral sclerosis, relapsing remitting MS, extrapyramidal symptoms, and late onset Alzheimer’s disease. All the patient groups displayed significantly elevated NF-L levels as compared to the controls. We also tested the assay’s ability to monitor the amount of axonal breakdown in an animal model of MS. The NF-L levels were found to be elevated in rodents with chronic experimental autoimmune encephalomyelitis, giving a possible tool for monitoring new treatment strategies for axonal protection in MS. When studying a large population based MS material, we found axonal breakdown to be present early in the disease course and the breakdown was observed both in active relapse and clinically stable disease, indicative of ongoing . NF-L levels were correlated to progression index, that is, high NF-L levels detected early in disease predict a fast progression of the disease. The amount of glial fibrillary acidic protein, a cytoskeletal protein found in , was also quantified and was shown to be a good marker for the more progressive MS subtypes, that is, primary progressive and secondary progressive disease, indicating formation of astrocytic scars and activation of astrocytes. The test dealt with in this thesis has the potential to identify the slow chronic degenerative diseases with progressive disappearance of nerve cells and their large myelinated axons. There is a significant need clinically to be able to quantify such types of degeneration in relation to the progressive disappearance of nerve functions and to relate these different conditions to treatment regimens, disease progress, and prognosis.

5 ABBREVIATIONS

Aβ Beta-amyloid AD Alzheimer’s disease AIDS Acquired immunodeficiency syndrome ALP Alkaline ALS Amyothrophic lateral sclerosis APC Antigen presenting cell CNS Central CREAE Chronic experimental autoimmune encephalomyelitis CSF Cerebrospinal fluid EAE Experimental autoimmune encephalomyelitis EDSS Expanded disability status scale ELISA Enzyme linked immunosorbent assay FTD GFAP Glial fibrillary acidic protein GFP Green fluorescent protein HLA Human leukocyte antigen HPLC High performance liquid chromatography HRP Horse radish peroxidase HSV-1 Herpes virus type-1 IFN Interferon mAb Monoclonal antibody MBP Myeline basic protein MHC Major histocompatibility complex MOG Myelin glycoprotein MRI Magnetic resonance imaging MS Multiple sclerosis MSA Multiple system atrophy NA Not applicable NF-H Neurofilament heavy NF-L Neurofilament light NF-M Neurofilament medium NFs Neurofilaments NPH Normal pressure hydrocephalus NSE Neuron specific enolase ON Optic neuritis OPCA Olivopontocerebellar atrophy PD Parkinson’s disease PI Progression index PNS Peripheral nervous system PPMS Primary-progressive MS PRMS Progressive-relapsing MS PSP Progressive supranuclear palsy RRMS Relapsing-remitting MS SAH Subarachnoid haemorrhage SOD Superoxide dismutase SPMS Secondary-progressive MS SWD Subcortical white-matter dementia T.b. gamb. Tryphanosoma brucei gambiense sleeping sickness TNF Tumor necrosis factor VAD Vascular dementia

6 INTRODUCTION

Neurodegenerative diseases

Degenerative diseases of the generally demonstrate slow, progressive loss of neurological functions in the patient without any apparent cause, for example, no infection, neoplasm, localized vascular disease, and toxicity [1, 2]. The diseases usually appear in middle age and later, but young individuals may sometimes be affected. Many of these diseases are acquired or sporadic, and some are inherited. The inherited forms may serve as models for the sporadic, more common forms. A few of these diseases are entirely inherited, for example, Huntingston's chorea [3-5]. The pathological changes seen within the nervous system and the anatomical location of the affected regions determine the clinical features of the diseases. Cerebral cortex afflictions impair the cognitive ability and could contribute to dementia. When the basal ganglia are involved, movement disorders may appear. Cerebellar and/or spinal diseases cause ataxia. Spinal disorders may also cause motor weakness. Neuronal loss and reactive gliosis can be observed in involved regions.

Alzheimer's disease (AD) is the most common degenerative brain disease, affecting the cerebral cortex. The incidence rises with age, and 30% of those over age 85 are diseased [6]. The appearence is sporadic and the etiology is unknown. Cerebral atrophy , senile plaques, and neurofibrillary tangles in the neocortex and limbic regions, all with amyloid accumulation and distorted can be observed [6-8].

The group of diseases which affect the basal ganglia, includes as the most common, Huntingtons chorea and Parkinson's disease. Huntington's chorea is

7 an autosomal dominant disease affecting 4, with an expanded CAG trinucleotide repeat expansion which causes degeneration of the nucleus caudatus, putamen and cerebral cortex. The prevalence of the disease in the USA and most European countries is approximately 5 cases in 100 000 individuals. The disease progress to dementia and death [3-5]. Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopaminergic in substantia nigra. The etiology of the disease is unknown but in α-synnuclein have been found in rare cases of familial PD [9]. The prevalence of the disease is about 1% of the population older than 60 years of age and the mean age of onset is estimated to be the early to mid 60s. The clinical manifestations include resting tremor, bradykinesia, and rigidity [10].

Amyothrophic Lateral Sclerosis (ALS), Friedreich's Ataxia, Multiple System Atrophy (MSA) belong to the group of diseases affecting the spinal chord and cerebellum, giving rise to degeneration and ataxia respectively. ALS is an adult-onset motor neuron disorder which is characterized by the death of motor neurons in the cortex, brain stem, and spinal chord leading to increasing weakness in the limbs and, ultimately, death from respiratory failure. Approximately 10% of ALS cases are familial, and the rest are sporadic and the prevalence is 4-6 per 100 000 individuals. It is estimated that 20% of the familial cases are owing to mutations in the superoxide dismutase (SOD) 1 [11-14]. Friedreich's Ataxia is an autosomal recessive cerebellar ataxia with a prevalence of 1-2 per 40 000 Caucasians and is caused by a GAA triplett repeat expansion in the gene for frataxin. The disease is characterized by posterior column degeneration and dentate nucleus atrophy [15-17]. MSA is a sporadic neurodegenerative disorder which generally arises after 60 years of age. The prevalence of the

8 disease is 2-5 cases per 100 000 individuals, and the disease is characterized by parkinsonism, cerebellar ataxia, and pyramidal signs [18, 19].

A significant group of are caused by generalized vascular disease. A single infarct in the cerebral artery is usually not sufficient to cause dementia, but multiple and bilateral infarcts or infarcts positioned in critical structures such as the and thalamus may elicit such symptoms. Vascular dementia is the second most common cause of dementia [20, 21]. There are two clinical conditions related to vascular dementia: multi-infarct dementia often a result of hypertension, and often with focal neurological symptoms, and the other type is caused by multiple emboli in the cerebral cortex. Also, infectious agents are able to induce dementia, for example Creutzfeld-Jacobs disease ( derived) [22, 23] and the AIDS-related dementia complex (HIV derived) [24-26]. Traumatic injuries may also cause dementia, such as accidents, and boxing [27-29].

Some neurodegenerative diseases affect several regions of the nervous system. Multiple sclerosis (MS) is characterized by multifocal regions of demyelination disseminated in space and time and distributed throughout the central nervous system. Multiple sclerosis is a demyelinating autoimmune disease. It is the most common cause of neurological handicap in young adults and usually develops between 20 and 40 years of age. It was estimated (2001) that the disease worldwide affects one million individuals. Women outnumber men by a factor of two, and the presence of HLA-DR and HLA- DQ increases the risk for developing the disease [30-32]. The disease typically initiates with a relapsing-remitting clinical course, characterized by one or more bouts followed by complete or partial recovery with a clinically stable disease. Within 10 years, 50% of the relapsing

9 remitting patients develop a secondary-progressive disease with a gradual progression of disability with or without relapses. A small percentage of the patients initially develops a progressive disease from the onset which can manifest itself with or without relapses, called primary-progressive and progressive-relapsing, respectively [30].

The procedure to diagnose neurodegenerative diseases starts with the careful evaluation of anamnesis and a physical examination. The clinical work up is aided by neuropsychological assessment of cognitive, conative, and emotional functions. There is an overwhelming body of scoring systems and scales to supplement this evaluation. Brain imaging techniques give essential information and MRI is valuable in assessing chronic brain disorders. Neurophysiological tests play a major role in diagnosis of motor neuron diseases and degenerative disorders of the peripheral nervous system. Furthermore, in the last decade analysis of the cerebrospinal fluid markers of neural tissue damage has gained increasing importance in the clinical setting.

Immunochemical considerations of brain-derived markers in CSF

Cerebrospinal fluid (CSF) surrounds the cells in the central nervous system and the major part of the protein content is of blood origin. The total volume of CSF present in a human at a given time is about 165 ml divided into ventricular (32 ml) and extra ventricular (133 ml) compartments. [33, 34]. CSF is constantly formed in the choroid plexus of the ventricles, and the daily amount produced varies with age, approximately 500 ml/day (0.4 ml/min) in young adults and 250 ml/day in the older population.

10 The CSF provides delivery of electrolytes, signaling molecules, and nutrients to the brain parenchyma as well as contributing to maintain a controlled physiological pH environment. Approximately 20% of the total protein content, which amounts to 1/10th of the plasma level, is brain- derived. A common feature of brain-derived proteins is their higher concentration in CSF compared to blood, inducing a net efflux of brain- derived proteins into the blood stream. The main exit pathway for CSF is the spinal subarachnoid spaces including the arachnoid villi and spinal nerve roots [33, 34]. The interstitial fluid surrounding the various cells of the central nervous system is in continuum with the cerebrospinal fluid. The release of cellular compounds into the during degeneration or acute damage of the CNS is thus reflected in the CSF.

Early diagnosis of degenerative brain diseases is important for medical intervention, and the lack of biochemical parameters to aid the clinical diagnosis emphasizes the need for diagnostic tests. The content of the cerebrospinal fluid surrounding the in the central nervous system may reflect the ongoing degeneration or the magnitude of acute damages.

Brain tissue contains several different cell types which express and sometimes release, into the cerebrospinal fluid, typically brain-associated antigens such as neurofilaments, glial fibrillary acidic protein (GFAP), S- 100, the Tau antigen (TAU), different forms of amyloid proteins (beta- amyloid), 14-3-3 protein, (MBP), and neuron specific enolase (NSE). These markers for brain damage are briefly described in figure 1.

11 Neurofibrillary Neuronal tangles Senile plaques damage Axonal P Tau β-amyloid NSE Axonal damage damage Tau NF Neuronal damage 14-3-3

Oligodendrocyte MBP

Glial damage S-100 GFAP

Figure 1. Brain Damage Markers.

Intermediate filaments The mammalian consists of three different filament systems: (25 nm), intermediate filaments (10-12 nm), and containing (7-10 nm). Intermediate filaments constitute approximately 1% of the total protein content in an average cell, but certain cells such as epidermal keratinocytes and neuronal cells can have an intermediate filament content of up to 85% [35].The intermediate filament family consists of more than 67 members divided into five different groups, based on their sequence similarities [36-38]. All intermediate filaments share a common structural scaffold consisting of a conserved α-helical coil region called the “rod”, flanked by a variable head and tail region [39, 40]. These filaments appear in the (type I-IV) with the exception of nuclear (type V) [41]. Assembly can occur in a heteropolymeric fashion between filaments from different sequence similarity groups. Table 1 presents the members within the intermediate filament group.

12 Table 1. The Intermediate Filament Family (Modified from [36]). Assembly Protein name Filament Number Tissue group type of genes distribution I Acidic I >25 All epitelia CK9-20 Basic II >24 All epitelia cytokeratins CK1-8 II III 1 Muscle cells GFAP III 1 Astrocytes/ III 1 PNS neurons III 1 Muscle cells III 1 Mesenchymal cells α- IV 1 CNS neurons Desmuslin IV 1 Muscle NF-L IV 1 CNS neurons NF-M IV 1 CNS neurons NF-H IV 1 CNS neurons IV 1 Heterogenous IV 1 Muscle III A/C V 1 Nucleus V 1 Nucleus V 1 Nucleus Orphan Phakinin Orphan 1 Filensin Orphan 1 Lens

Neurofilaments Neurofilaments (NFs) belong to the intermediate filament family and constitute the major cytoskeletal components in neuronal cells. They are of importance for maintaining the structural integrity and calibre of the axons and dendrites and thereby the conduction velocity of nerve impulses [42- 44]. There are three neurofilament chains which are classified owing to their sizes as determined by SDS-page, that is NF-L (68kDa), NF-M (150 kDa), and NF-H (200 kDa) [45, 46]. The expression of neurofilaments are developmentally regulated with NF-L expressed early in neuronal differentiation followed by the expression of NF-M at the emergence of

13 formation, whereas NF-H is expressed late in neuronal differentiation during postnatal development [47, 48].

Several knockout studies have been conducted in which single neurofilament chains have been targeted and it seems that the protein is non essential for neuronal development and function [42, 49, 50]. The filaments are obligate heteropolymers in vivo in which NF-L forms the backbone to which the heavier chains copolymerize to form the 10 nm neurofilament fibre [45, 51-54]. Neurofilament light is the quantitatively most common filament with a molar ratio of 4:2:1 (NF-L, NF-M, NF-H) [55].

Neurofilaments have a structural conserved core region, the “rod” characteristic for the intermediate filament family with variable C- and N- domains. The core forms the structural unit for filament assembly [56]. The C-terminus is extended in NF-M and NF-H, forming so called “side arms” believed to form crossbridges between neurofilament fibres [57, 58]. NF-M and NF-H are among the most phosphorylated proteins in the nervous system with approximately 108 phosphate groups on NF-H and 24 on NF-M respectively, NF-L on the other hand has only three sites [59]. Phosphorylation of neurofilaments has a negative impact on rate and is belived primarily to be a result of extensive phosphorylation of the C-terminus of NF-H [60-62]. Neurofilaments are synthezised in the and are transported into the towards the end of the axon. It has been shown by GFP labeling of NF-H that the filaments are transported both in an anterograde and retrograde fashion. The net transport is however anterograde with an average maximum velocity of 1.3 µm/sec with intermittent pauses implying slow axonal transport [63]. Neurofilament breakdown is mediated by calcium dependent in

14 the family [64, 65]. Calpain family members present in neurons are of two types, type I and type II. They differ mainly in the amount of calcium needed for optimal activation in vitro. Calpain I (µCalpain) needs micromolar amounts of calcium, whereas calpain II (mCalpain) demands millimolar amounts [66]. Mutations in the NF-H gene have been found in a small number of ALS cases, and mutations in the NF-L gene have been found to be the cause of Charcot-Marie-Tooth type 2E neuropathy [67-69]. A single finding of a in the NF-M gene has been found in early onset PD [70].

Glial fibrillary acidic protein Glial fibrillary acidic protein (GFAP) is the most abundant intermediate filament protein in mature astrocytes. It is a cytoskeletal protein which is thought to be important for modulating and shape by stabilizing the astrocyte extensions. GFAP is a classical marker of astrogliosis, known as the scar formation obtained by astrocytes adjacent to injuries in the CNS [71, 72]. Increased levels have been found following stroke, , and various neurodegenerative brain diseases [73-77].

Non intermediate filament markers

S-100 S-100 belongs to a large family of EF-hand proteins. The protein was originally characterized as a group of small acidic proteins enriched in nervous tissue. The S-100b protein has a molecular size of 21kDa and contributes to cell to between astrocytes and neurons, transcellular , and also maintenance and development of

15 the CNS. It is mainly found in astrocytes, Schwann cells, , tissue, and adipocytes [78-80]. S-100 has been found to be elevated in patients with stroke, cerebral haemorrhage, and central nervous system malignancies [73, 81, 82]. S-100 is used worldwide to monitor brain injuries [83] and can also be used as a owing to its presence in melanocytes and [84].

Tau Tau is found in neuronal axons. There are six different isoforms of tau with molecular weights between 50 and 70 kDa. Tau is a associated protein and is believed to be important in microtubule assembly and stabilization [85, 86]. Elevated tau levels have been found in head trauma patients, Alzheimer’s disease and in patients with relapsing-remitting Multiple Sclerosis [87-89]. Deposition of hyperphosphorylated tau is also a prominent feature of neurofibrillary tangles found in dementias such as Alzheimer’s disease [90, 91].

β-amyloid β-amyloid (Aβ) is 4kDa protein produced from proteolytic of the amyloid precursor protein (APP) found in senile plaques. The protein has been shown to be toxic for neurons and is belived to be the major hallmark of Alzheimers disease, together with phosphorylated tau in neurofibrillay tangles. β-amyloid is found in CSF from patients with Parkinson’s disease, progressive supranuclear palsy, and various dementias such as dementia. Patients with Alzheimers disease have a 50% decrease in β- amyloid levels as compared to controls [92-94].

16 14-3-3 proteins There are 7 different isoforms of 14-3-3 proteins with an average monomeric molecular size of 30 kDa. The proteins are abundantly expressed in neuronal cells and are involved in signalling processes. The proteins are normally localized to presynaptic vesicle membranes and synaptic membranes but are also found in non nervous tissue such as liver and intestine [95, 96]. Highly elevated levels of 14-3-3 have been found in the CSF from patients with Creutzfeldt-Jakob disease [97, 98].

Myelin basic protein Myelin basic protein (MBP) is a major constituent of myelin and approximately 30% of the CNS myelin protein and 18% of PNS myelin is MBP. Myelin basic protein is an 18.5 kDa protein localized to neurons, neuroendocrine cells, erytrocytes, central and peripheral myelin, and developing oligodendroglia [80, 99]. It has been shown to increase in the CSF in MS patients with an active relapse [100].

Neuron specific enolase Enolase is a glycolytic enzyme and exists in α-,β-, and γ-forms. The γ-form is called neuron specific enolase (NSE) and has been found in neurons, neuroectodermal, cells and erytrocytes. [80, 101, 102]. The protein is mainly located in neuronal cell bodies, and it is also present in axons. Increased levels of NSE have been found both in serum and CSF following acute injury such as stroke, seizures, and brain infectious diseases [74, 81, 101, 103, 104].

17 Consequences of degenerative or traumatic brain disorders

Following brain damage, these brain associated proteins can be released into the cerebrospinal fluid (CSF) and may reach the blood circulation. Such damages include stroke, traumatic brain lesions, concussions following accidents, and several neurological diseases of the brain. It is possible that quantification of brain-associated proteins in blood or CSF can be related to the degree of damage or disease. For some of these markers, assays are available.

Acute lesions in the central nervous system cause leakage of proteins from different nerve cells to the cerebrospinal fluid and serum. By measuring the amount of these proteins in the cerebrospinal fluid, an estimate of the degree of damage can be obtained. Following focal damages within the central nervous system, these analyses support other diagnostic approaches such as radiological. However, following diffuse non-focal damages, engaging large parts of the brain, such as encephalitis, postanoxic encephalopathy, neonatal asphyxia, and cerebral vasculitis, no focal changes can be seen by traditional neuroimaging techniques and the extent of brain damage has to be evaluated simply by observing the patient and performing ordinary clinical examination.

Different degenerative processes in the brain are being subjected to significant interest owing to the increasing mean age in the western world. Such diseases include amyotrophic lateral sclerosis (ALS), vascular type dementia, frontal lobe dementia, progressive supra-nuclear paralysis, multiple system atrophy, low pressure hydrocephalus, and multiple sclerosis (MS). The test dealt with in this thesis has the potential to identify these

18 slow chronic degenerative diseases with progressive disappearance of nerve cells and their large myelinated axons. There is a significant need clinically to be able to quantify such types of cell degeneration in relation to the progressive disappearance of nerve functions and to relate these different conditions to treatment regimens, disease progress, and prognosis.

Damages or diseases within the nerve system, which cause nerve cell death may lead to either necrosis or [105-107]. The degree of damage affects the mechanism which will be activated. Profound damages are dominated by necrosis including destruction of cell membranes, collapse of the cell morphology, release of intracellular molecules including S-100, and activation of proteolytic leading to . S-100, which is an intracellular calcium binding protein, is a very useful marker for rapid traumatic brain damages owing to this mechanism [108, 109]. Apoptosis, which usually appears after more diffuse brain damages, is actively initiated and geared by certain genes. It is characterized by a morphologically reduced , fragmented cytoplasma, and uptake of remaining cell constituents by cells nearby with no apparent sign of inflammation. The substances which may be released can be more difficult to detect since they are rapidly reabsorbed. Both these processes can occur simultaneously.

Animal models of neurodegenerative diseases

Several of the neurodegenerative diseases can be modelled in and many genetical mice models are currently available such as spinal muscular atrophy [110], Huntington’s disease [111], Amyotrophic lateral sclerosis [112], Parkinson’s disease [113], Friedereich’s ataxia [114], and Alzheimer’s disease [115]. Neurodegenerative diseases can also be induced by immunization with brain specific proteins. Experimental autoimmune

19 encephalomyelis (EAE), is an animal model of MS. This model was developed in 1933 using monkeys immunized with rabbit brain emulsions [116]. EAE can be induced in various species but the most common is rodents. Some models involve an acute monophasic disease wherease others develop a chronic relapsing-remmiting subtype [117, 118]. A chronic relapsing-remitting disease type, chronic experimental autoimmune encephalomyelitis (CREAE) can be induced in rats by immunization with myelin oligodendrocyte glycoprotein (MOG), is associated with axonal damage in active and inactive demyelinating lesions, and is believed to be the model with the closest resemblance to MS [119-121].

Table 2. Comparison between EAE disease models and MS (Modified from[122]). EAE MS Etiology Myelin auto- Unknown antigens and antigen-specific T cells Genetics MHC-Linked susceptibility Yes Yes Female predominance Yes Yes Clinical manifestations Disease course Monophasic or Relapses and chronic chronic relapses progressive Paralysis Yes Yes Ataxia Yes Yes Visual impairment Yes Yes Immunology T cells reactive to myelin Yes Yes Antibodies to myelin Yes Yes TNF-α, IFN-γ Increased Increased Demyelination Yes (some models) Yes Axonal damage Yes (some models) Yes

20 AIMS OF THE THESIS

• To establish a panel of highly discriminatory neurofilament light mononclonal antibodies. • To generate a highly sensitive two-site non-competitive for measurements of neurofilament light levels in cerebrospinal fluid. • To validate the ability of the immunoassay to detect elevated levels of neurofilament light in different degenerative neurological diseases. • To adapt the immunoassay to monitor neurofilament content in rat cerebrospinal fluid at experimental autoimmune encephalomyelitis. • To evaluate the clinical relevance of neurofilament light and glial fibrillary acidic protein as biochemical markers of Multiple Sclerosis.

21 METHODS

Generation of monoclonal antibodies

Monoclonal antibodies are usually produced by immunizing mice with the antigen of interest in Freunds complete adjuvant. In a humoral immune response to a T-cell dependent antigen, antigen presenting cells (APC) (dendritic cells) take up the antigen and process it into which are presented on MHC class II molecules on the cell surface. The appropriate T Helper cells recognize the on the APC and are activated to proliferate and differentiate into a cytokine producing state. At the same time B-cells bind the antigen independent of the T-cell on its surface through the B-cell receptor. The antigen is taken up and processed by the B- cell in a similar fashion as the APC, and the T-cell epitope is expressed on MHC class II molecules on the surface. The activated T helper cells can now recognize T-cell epitopes of the nominal antigen on the surface of the B-cell, interact with the B-cells and provide the additional signals needed to drive the B-cells to clonal expansion and antibody-producing plasma cells [123]. Such individual plasma cells are fused to myeloma cells according to Köhler and Milstein 1975 [124] and significant amounts of antibody can be produced. It should however be stressed that not all antibodies produced are useful for biochemical investigations. The antibody needs to bind with high affinity to its antigen and the binding should optimally be highly specific for the target.

Enzyme linked immunosorbent assay

Enzyme linked immunosorbent assay (ELISA) is an immunoassay which uses antibodies as tools to detect and/or quantify various biological molecules in solution and was developed in the beginning of the 70s [125].

22 The most commonly used assay for measuring substances in fluids is termed the “two-site” or “sandwich” ELISA which uses a solid support upon which a catcher antibody is immobilized and a tracer antibody which can recognize another epitope on the antigen different from what is reactive with the catcher antibody [126]. When binding of the tracer antibody has occurred, an enzymatically conjugated detection antibody can be added, and with the addition of substrate, the absorbance of a coloured product is measured. There are numerous different enzymes used for detection in ELISA and the most common ones are horse radish perioxades (HRP) and (ALP). We have chosen to use a commercially available enhancement substrate AMDEX from Amersham Pharmacia Biotech which has the advantage of incorporating multiple HRP enzymes in the detection of the secondary antibody yielding a 10-100 fold increase in signal intensity. The setup of our assay is in figure 2.

Step 1 coating Step 2 add Step 3 add Step 4 add Step 5 add

Coating antibody mAb 47:3 Antigen CSF containing NF-L Tracer antibody biotinylated mAb 2:1 Conjugate SA-HRP (Amersham) Substrate OPD

Figure 2. Assay Set-up.

23 RESULTS

Paper I: Norgren N, Karlsson JE, Rosengren L, Stigbrand T. Monoclonal antibodies selective for low molecular weight neurofilaments. Hybrid Hybridomics. 2002 Feb;21(1):53-9.

Bovine and macaque NF-L were purified by HPLC using MonoQ HR column chromatography. The purified antigens were used as immunogens for the production of monoclonal antibodies according to Köhler and Milstein 1975 [124]. Six hybridomas out of 100 were chosen for further antibody production. No major cross-reactivity against other brain antigens, that is, GFAP, S-100, MBP, NSE, NF-M, and NF-H was detected using ELISA. Three out of six antibodies displayed non-overlapping epitopes as proven by V8-protease degradation in consert with sequential and pair-wise BIAcore epitope evaluation. Four independent epitopes were identified and defined by the individual mAbs — 12C2, 34B2, and 47:3 and as well as a cluster of mAb — 2:1, 43C2, and 45.

Paper II: Norgren N, Rosengren L, Stigbrand T. Elevated neurofilament levels in neurological diseases. Brain Res. 2003 Oct 10;987(1):25-31.

By semi-quantitative affinity determination, mAb 47:3 and 2:1 were selected as the components in the two-site assay. The assay has the ability to detect 60 ng/l NF-L and the standard curve ranges from 60 to 64 000 ng/l. Variation within plates as determined by coefficient of variation was 0.06

24 and between plates was 0.09. The ability of the assay to quantify NF-L in CSF was evaluated by measuring protein levels in patients with neurological disease. Only 2 out of 11 healthy controls demonstrated neurofilament levels above the cut off level of 100 ng/l. All patient groups examined, that is, cerebral infarction, amyotrophic lateral sclerosis, relapsing-remitting multiple sclerosis, extrapyramidal symptoms, late onset Alzheimer’s disease, and vascular dementia had significantly elevated NF-L levels as compared to controls.

Paper III: Norgren N, Stigbrand T. Cerebrospinal fluid levels of neurofilament light in chronic experimental autoimmune encephalomyelitis. Submitted to Brain research Bulletin.

The two-site ELISA assay was adapted to measure neurofilament light levels in rat CSF. Chronic experimental autoimmune encephalomyelitis was induced in Dark Agotti rats by immunization with rat MOG (aa 1-125). All immunized rats developed EAE and the disease course was followed and evaluated using EAE scoring. The CSF was aspirated from the Magna of anaesthetized animals and the NF-L content was estimated. We found a significant increase in NF-L levels in immunized animals as compared to controls.

25 Paper IV: Norgren N, Sundström P, Svenningsson A, Rosengren L, Stigbrand T, Gunnarsson M. Neurofilament and glial fibrillary acidic protein in multiple sclerosis. Neurology, 2004. 63(9): p. 1586-1590.

Neurofilament and GFAP levels were quantified in 99 patients with different subtypes of multiple sclerosis. Both NFL and GFAP levels were elevated in MS patients as compared to controls. Presence of relapse yielded significantly higher NFL levels while GFAP was unaffected. We found a positive correlation to prognostic index (PI) both in patients with an active relapse (r=0.49, p<0.01) and clinically stable patients (r=0.29, p<0.05). A strong correlation was detected between NFL levels and inflammatory cell counts in patients with an ongoing relapse (r=0.52, p<0.001). All but one of the controls had NFL levels below the detection limit, and the highest value observed in a MS patient was as high as 4500ng/L. All three major subgroups of MS patients, i.e. RR, SP and PP MS presented highly elevated levels, with the highest levels found in patients which developed a SP disease within the follow up period of this study.

DISCUSSION

The new marker, the neurofilament protein “neurofilament light”, has so far not been possible to quantify in a reproducible manner using monoclonal antibodies. One of the crucial aspects of developing highly sensitive and specific immunoassays is the generation of the antibodies involved in the

26 tests, which have to be extremely selective in terms of specificity for the assembled, processed and post-translationally modified antigen. The uniqueness in specificity of the antibodies identifying different forms of the antigens and the possibility to relate serological results to different disease states are crucial.

Production and characterization of anti NF-L antibodies

We were able to generate six monoclonal antibodies highly specific for neurofilament light [127]. We chose to use antigens from both bovine and macaque source. The Macaca family of monkey belongs to the old world monkeys. They have a 93% genomic sequence similiarity to humans [128] and is likely to display a more human-like neurofilament light protein than would the bovine counterparts. Enzymatic degradation with V8 protease was carried out as well as serial and pairwise epitope mapping using BIAcore and both techniques pointed to four independent epitopes. The antibodies were produced from both bovine and macaque sources. All antibodies were shown to react with the conserved α-helical rod region consisting of 308 amino acids [56]. We tested the antibody cross reactivity to other common brain antigens from different cells of the nervous system such as the neuronal protein NSE and the glia protein MBP, as well as the astrocytic proteins GFAP and S-100, and the other neurofilament chains, that is, NF-M, and NF-H. No major unspecific binding was found. Two of these antibodies were outstanding in their performance in binding the antigen, that is, mAb 2:1 and 47:3, and they were choosen as components in the development of our immunoassay for neurofilament. These monoclonal antibodies were both obtained from immunization with monkey antigen stressing the importance of close resemblance of the species of interest and the source of antigen.

27 Establishment of a highly discriminative NF-L ELISA

ELISA is the most common assay for measuring soluble proteins in plasma. There are currently a large number of commercially available assays for different antigens, and ELISA has almost completely replaced the use of radioimmunoassays owing to lower cost and the fact that the handling of radioactive material can be potentially dangerous. The sensitivity of ELISA assays can be significantly enhanced by the use of substrates which can incorporate multiple enzymes for the conversion of the development substrate.

Our assay is capable of measuring minute amounts of NF-L in CSF, and we found elevated levels of NF-L in all neurologically diseased patient groups tested [129]. We found the highest levels of neurofilament light in patients with cerebral infarctions (19 800 ± 9 100 ng/l) which reflect the massive damage to neuronal tissue. It has previously been shown that neurofilament light measurements in the CSF can detect small infarctions [130]. Patients with amyotrophic lateral sclerosis also displayed highly elevated NF-L levels (3 600 ± 1 200 ng/l) as expected, since the pathology of this disease mainly affects large motor neurons connecting nerve impulses from the spinal chord to the muscles [131]. The large difference in NF-L levels might be explained in part by the heterogeneity of the disease with patients suffering from upper or signs. signs involving the pyramidal tract are associated with highly elevated CSF NF-L levels [130]. Neurofilaments are mainly localized to the axons of myelinated tissue, and elevated levels in the CSF therefore are mainly indicative of axonal damage. Patients with vascular dementia and extrapyramidal symptoms displayed moderately elevated NF-L levels, 1 400

28 ± 800 ng/l and 1 100 ± 300 ng/l respectively. In patients with vascular dementia, the subcortical white matter is most often affected with lacunar infarctions, axonal breakdown, and myelin changes. The patients with extrapyramidal symptoms represent a heterogenous group of patients with Parkinson’s disease (PD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA). All these diseases involve the basal ganglia. The patients with Parkinson’s disease all displayed low levels of NF-L in the CSF, whereas PSP and MSA patients displayed a 9.5 times higher level of neurofilament light. It has previously been shown that measurements of neurofilament light can be used to differentiate PD from PSP and MSA cases [132]. We found a small increase of NF-L in the CSF of patients with late onset Alzheimer’s disease (LAD); 300 ± 100 ng/l. Alzheimer’s disease pathology is dominated by degeneration of cortical neurons and the white matter damage is less severe. Thus, any significant increase in neurofilament light levels was not expected. In analyzing the patients with relapsing-remitting multiple sclerosis, we found a large increase in NF-L levels: 2 500 ± 1 500 ng/l, indicating large axonal damage. The disease has been regarded as an autoimmune disorder mainly affecting the myelin sheath covering the neurons, but the involvement of axonal damage has recently been stressed as an important factor in the pathology [133-135].

Neurofilament light measurements in an animal model of MS

Since NF-L is a marker of mainly axonal damage, we questioned if elevated levels of the protein could be detected in an animal model of multiple sclerosis with known axonal damage. Rats with EAE induced with recombinant rat MOG (aa 1-125) displayed significantly elevated levels of

29 NF-L in the CSF as compared to controls, thus indicating that direct axonal damage is taking place [136]. A redistribution of N-type calcium channels in injured axons has been found in EAE and MS, and this might increase the intracellular Ca2+ and thereby trigger the activation of leading to neurofilament breakdown [137]. Calpain translational activity is highly increased (206.5%) in rats with EAE, and the protein level in the spinal chord decreases (42.9%) which further strengthens the hypothesis of axonal damage [138]. We suggest that NF-L can be used as a marker of neuronal damage in rats with EAE and can be a most valuable indicator in validating new treatment strategies of the disease.

The main current immunomodulatory treatment for MS is interferon-β1 and glatiramer acetate which both work by reducing the frequency of boots in relapsing remitting MS. Type 1 interferons are cytokines which are believed to bias the proinflammatory Th1 cells to the anti-inflammatory Th2 type of responses. Glatiramer acetate is a standardized mixture of synthetic peptides of L-, L-alanine, L-, and L-, and it is thought to induce tolerance to MBP specific T-cells and to change the immune response from a Th1 to a Th2 response [31, 139]. The treatment by both drugs has been evaluated in EAE models, and future studies of novel compounds are most likely to be made in a rodent system prior to human pilot studies.

MS and prognostic impact of brain-marker levels in CSF

We chose to further explore the prognostic value of measuring biomarkers in the CSF of patients with MS. A prospective study of all available MS cases in Västerbotten County from 1988-97 was made, and NF-L and GFAP levels were quantified and compared to paraclinical data [140]. The setup of

30 the study was that the CSF was collected early in the disease progression and a clinical follow up was conducted at a later time (average time after collection, 4 years). Since the two markers measure two different processes, that is, axonal degeneration and astrogliosis, it would be of interest to see if these processes could be monitored separately in the different disease types. GFAP displayed the highest levels in secondary and primary progressive MS in which a prominent astrogliosis would be expected. Secondary and primary progressive MS types are characterized by progressive disease type with or without any presence of relapses and a relatively more aggressive disease [141]. A significant elevation of NF-L was found in patients relapsing within 3 months before sample collection as compared to clinically stable patients. When separating patients with recent relapses from stable subjects, we found a statistically significant increase in NF-L in SP and RRMS patients as compared to controls (p<0.001 and p<0.01 respectively) with a trend of higher levels in patients with SPMS. Owing to the higher level of neurological deficit in SP MS, a more pronounced axonal damage is not unexpected [142].

It has been postulated that axonal alterations are present in patients with MS. Trapp and coworkers [143] found axonal ovoids intensely stained for neurofilaments in patients with SP and PPMS, which is indicative of axonal transection. More than 11 000 axonal ovoids/mm2 were found in active lesions, 3000 at the edges of chronic active lesions, and 800 in the center of chronic active lesions as compared to less than 1 in white matter of the controls [143]. It has been shown in animal models that demyelination causes decreased neurofilament phosporylation and reduction of the axonal caliber [144]. The phosphorylation of neurofilaments is known to protect

31 them from degradation, and therefore demyelinated tissue might be more prone to axonal neurofilament breakdown.

To investigate the relevance of making prognostic predictions by quantifying GFAP and NF-L, we calculated a prognostic index (PI) defined as expanded disability status scale (EDSS)/disease duration [145, 146]. PI might be seen as a way of estimating the rate of progression and thereby the severity of disease. Both the neuronal and astrocyte marker displayed a positive correlation to prognostic index. The strongest correlation was found for NF-L in patients with an active relapse indicating that a high amount of NF-L in the CSF early in the disease predicts a more rapid progression of the disease. There are indications of axonal damage in normal appearing white matter [135, 147], and the presence of amyloid precursor protein indicative of early axonal damage has been reported [148]. We furthermore compared the levels of NF-L and GFAP to the inflammatory cell count, and a highly significant correlation between NF-L levels and cell count was found in RRMS patients with an active relapse (p=0.001, Spearman’s ρ=0.52). It is known that an active relapse is correlated with increased inflammatory activity. A strong correlation between neurofilament levels and inflammatory cell number shows that axonal damage might be related to the inflammatory process in acute lesions. Our results also indicate that axonal damage is present both during the presence and absence of relapses indicating an ongoing neurodegeneration.

Brain proteins as biomarkers of neurological diseases

Given the large number of neurodegenerative diseases present in the population, the need for a good diagnostic and/or prognostic tool is of utmost importance. NF-L seems to be a good marker for axonal damage in

32 both traumatic and slow degenerative disease. Neurofilament inclusions are a common feature in diseases affecting the nervous system. Perikaryal and proximal axon accumulation of neurofilaments are often found in patients with ALS [11]. Lewy bodies are common feature in PD and related disorders and have been shown to be immunopositive for neurofilaments [149]. Neurofilament has also been found in AD and dementia with Lewy bodies [149-151]. A newly diagnosed disease called neurofilament body disease, characterized by dementia and a rapidly progressive disease course, displays a large cytoplasmic deposition of neurofilaments [152, 153]. The direct involvement of neurofilaments in disease can not be ruled out and their leakage from affected cells can further increase the concentration in the vicinity of the neuron and ultimately the total CSF level.

To date five different immunoassays for the detection of neurofilament proteins in the CSF have been presented, and the majority of these assays have been studying NF-L (Table 2.). A recent assay has been constructed using commercially available antibodies for the phosphorylated form of NF- H. The highest levels of NF-H were found in patients with subarachnoid haemorrhage (SAH) followed by patients with ALS, space occupying lesions, and disc prolapses [154]. In another study using the same immunoassay, significantly elevated levels of phosphorylated NF-H were found in patients with optic neuritis [155]. One major drawback of this assay is that it is specific for only the phosphorylated form [156] so there might be large quantities of unphosphorylated NF-H present which remain undetected. One interesting finding is that the phosporylated form of NF-M and NF-H increases with age [157]. Since it is known that phosphorylation of neurofilaments [60-62] decreases the axonal transport rate, it might have

33 a negative effect on the plasticity and conduction velocity of aged neurons. Increase in CSF NF-L levels in patients with MS has been detected in three different assay systems [129, 130, 158]. A discrepancy between the assay systems still exists; Semra and coworkers [158] obtained the highest values in patients with PPMS whereas the two other assays recorded the highest levels in RRMS patients with an active relapse [140, 159]. This might in part be explained by the methodological differences between ‘dot-blot’ and ELISA, and also Semra had a low number of RRMS patients with no record of the presence or absence of active relapse. It is known that patients with an active relapse have significantly higher NF-L levels than the clinically stable patients [140, 159]. Slow neurodegenerative diseases suchs as AD, ALS, PD, and various other dementias all give rise to significantly elevated CSF levels of neurofilaments [130, 157, 160-163]. In a study made on patients with subcortical white-matter dementia, a sensitivity of 85% and a specificity of 68% was obtained by a NF-L cut-off of 442 ng/ml to differentiate between the diseased and neurologically healthy controls [163]. When comparing the CSF levels of NF-L in a group of patients with mixed dementias, that is, VAD, AD, and FTD, to healthy controls, the demented under age 65 were differentiated from the healthy with a specificity of 94% and a sensitivity of 82% [161]. Neurofilament determinations have also been used to differentiate between different Parkinsonian syndromes, that is, to predict clinical PD from MSA or PSP. The sensitivity to identify the non- AD group was 78% and the specificity was 80% on the basis of NF-L CSF determinations, and the combined test measuring NF-L and levodopa response gave a sensitivity of 94% and specificity of 88% [160]. When looking at NF-L levels to predict poor outcome after cardiac arrest, a strong correlation was found between anoxia time and coma depth, and a cut off level of 9 600 µg/l gave a negative predictive value of 90%. This means that

34 the majority of patients exceeding this CSF level for NF-L has a poor outcome prognosis [164]. Even diseases not traditionally looked upon as primarily of neurological nature, such as AIDS and sleeping sickness, have been shown to display elevated levels of NF-L indicative axonal destruction [165, 166].

35 Table 2. Neurofilament immunoassays (for abbreviations see page 6).

Specificity Assay Antibody Standard range Disease Reference type Phosporylated Two site 1º ab mAb SMI35 200-2.000 ng/l SAH [154] NF-H ELISA (Sternberg monoclonals) ALS [154] 2º ab pAb rabbit anti Space [154] NFH (Affinity research) occupying 3º ab HRP-swine anti lesions rabbit (DAKO) Disc [154] prolapses ON [155]

NF-L Two site 1º ab hen anti NF-L 125-16.000 ng/l ALS [130] ELISA (Inhouse) AD [130, 161, 162] 2º ab rabbit anti NF-L VAD [130, 161] (Inhouse) SWD [163] 3º ab HRP-donkey anti FTD [161, 162] rabbit (Amersham) MSA [160] PSP [160] PD [160] OPCA [130] NPH [130] Cerebral [130] infarction Cerebral [75] vasculitis MS [159, 167] AIDS [165] HSV-1- [104] encephalitis T.b. gamb. [166] Cardiac arrest [164]

NF-L Two site 1º ab mAb 47:3 (Inhouse) 60-64.000 ng/l ALS [129] ELISA 2º ab mAb biotinylated AD [129] (Our 2:1 (Inhouse) VAD [129] assay) 3º ab SA-HRP MSA [129] (Amersham) PSP [129] PD [129] Cerebral [129] infarction MS [129, 140] CREAE [136]

Phosporylated Two site 1º ab R61d rabbit anti NA AD [157] and non- ELISA NF-L/M/H VAD [157] phosporylated 2º ab mAb SMI31, NF-M/H SMI32, SMI33, SMI34 NF-L anti NF-M/H (Sternberger Inc.) mAb NR-4 anti NF-L (DAKO) 3º ab ALP goat anti mouse (Jackson)

NF-L Dot blot 1º ab mAb anti NF-L NA PPMS [158] (Serotec) 2º ab HRP-rabbit anti mouse (Sigma)

36 The field of biological markers for neurodegenerative diseases is expanding.

Total tau and Aβ1-42 are the most commonly used CSF markers for AD. The specificity for total tau is 90% and a mean sensitivity 81% (2500 AD cases,

1400 controls) and for Aβ1-42 the specificity is 90% and sensitivity to discriminate AD from normal aging is 86% (600 AD cases, 450 controls) [92]. Detection of protein 14-3-3γ is a common diagnostic criterion for CJD and a recent publication [98] has reported a sensitivity of 82% and specificity of 94% for sporadic disease when using a capture assay for detection which is comparable to the results obtained by the most commonly used method, ‘western-blot’.

S-100 and NSE measurement in the CSF and blood are hampered by the fact that these markers are not nervous system specific but have shown to be useful measures of brain injuries and for prediction of outcome [74, 109, 168]. It has furthermore been shown that S-100B and NSE levels are significantly correlated to lesion size in stroke patients [81]. GFAP has been shown to correlate to the neurological deficit as determined by EDSS in patients with SPMS [159], and a slight association has been shown to PI in the total cohort of patients with MS [140]. Highly elevated values of GFAP have been found in patients with cerebral infarctions and subarachnoid hemorrhage [77, 169], and the presence of highly increased GFAP amount predicts poor outcome in severe traumatic brain injuries [74].

Neurofilament detection can be used in wide variety of neurological diseases where axonal damage can be expected, and by selecting an appropriate cut off level, differential diagnosis between different diseases can be made [160]. It is known that neurofilament content is increased in the CSF with age [157, 161], and neuronal and axonal breakdown may

37 represent a subclinical pathological process involving white matter changes with an increasing prevalence with age. One obvious future use of the assay is to monitor novel treatment strategies aimed specifically towards axonal protection in neurodegenerative diseases. In the case of MS, given the known aspect of early axonal destruction, the need for drug intervention to retard the breakdown of neuronal tissue is urgent. It is of further interest that NF-L is positively correlated to progression index in patients with an active relapse. When an effective treatment becomes available, those patients with high levels of NF-L in the CSF early on in the disease should be treated immediately in order to minimize the neurological deficits.

For the first time we have been able to generate a highly sensitive and specific immunoassay for the detection of NF-L in the CSF. The assay is based on the use of two different monoclonal antibodies with a commercially available enhancement step giving an unlimited amount of reagents and a high reproducibility. We believe that this assay will be an important tool for further studies of neurodegenerative diseases. So far it has not been possible to measure neurofilaments in body fluids other than the CSF. The ability of measuring neurofilament in serum would make the test even more useful since lumbar puncture always is associated with a certain risk of injury and the availability of CSF samples is limited. Our immediate future goal is to adapt the assay to serum measurements of NF-L.

38 CONCLUDING REMARKS The main findings made in this thesis can be summarized as follows:

• Six highly specific monoclonal antibodies against macaque or bovine NF-L were generated. Four independent epitopes were elucidated using Biacore evaluation and V8 protease degradation. Individual mAbs — 12C2, 34B2, and 47:3 and the cluster of mAbs — 2:1, 43C2, and 45 are reactive with these epitopes.

• Monoclonal antibodies 2:1 and 47:3 were chosen components in a two site ELISA assay for detection of NF-L in body fluids because of their outstanding ability to bind the antigen.The assay has a least detectable dose of 60 ng/l and the standard range 60 to 64 000 ng/l.

• We were able to detect elevated levels of NF-L as compared to controls in a wide variety of patients with neurological diseases (cerebral infarction, amyotrophic lateral sclerosis, relapsing remitting multiple sclerosis, extrapyramidal symptoms, and late onset Alzheimer’s disease.)

• NF-L levels were found to be elevated in the CSF of animals with MOG induced EAE further strengthening the resemblance between MS and EAE and giving a possible tool for monitoring novel treatment regimes for axonal protection in MS.

• Axonal breakdown is present early on in MS patients, and breakdown is observed both during active relapse and clinically stable disease, indicative of an ongoing neurodegeneration. Neurofilament levels are correlated to prognostic index which predicts a rapid worsening of disease when high levels are obtained early in the disease progression.

• GFAP was shown to be good marker for the more progressive MS subtypes, primary progressive and secondary progressive disease, where astrogliosis is a more pronounced phenomenon. Combining both markers gives clues to two important active processes in MS pathology, namely axonal breakdown and formation of astrocytic scars.

39 ACKNOWLEDGEMENTS There are a number of people I would like to express my gratitude to:

My supervisor prof. Torgny Stigbrand for giving me the opportunity to conduct research and the helpful and positive attitude which you always display no matter if you are busy with other things or not. During the years we have spent some time together, equipped with our fishing rods trying to catch the “large” fish. And I would like to say that you still are the king of “spigg”.

Dr Lars Rosengren for being such a great collaborator and all the “clinical” advice.

David Eriksson for being such a good friend – I’m still envious on the Hälleflundra you caught in Norway, but I’ll guess there will be more opportunities to catch something bigger. Ali Sheikholvaezin and Homa Mirzaie for help with experiments. Martin Gunnarsson for being a good friend, and the fruitful collaboration we had – good luck with your future research in Örebro.

Berith Nilsson, Lisbeth Ärlestig, Amanda Ullén and Per Sandström for helping me to get started back in the “old” days.

Prof. Sten and Marie-Louise Hammarström and their former and present Ph.D. students and technical personnel, Anna Bas, Anna Fahlgren, Göte Forsberg, Arman Rahman, Anna Ildgruben, Marianne Sjöstedt, Anne Israelsson and Elisabeth Granström for scientific advice and pleasant discussion during the coffee breaks.

Dr Vladimir Baranov for scientific advice and all the interesting discussions we have had during breaks.

Lena Hallström-Nylén and Birgitta Sjöberg for making everyday life at the department easier.

Susan Jeglum for the linguistic correction of the thesis.

Teknikbrostiftelsen and the Medical faculty of Umeå University are acknowledged for financial support.

40 Till alla roliga och trevliga människor som jag umgåtts med på fritiden: Tommy, Johanna, Jeanette, Sofia, Magnus, Martina, Erik, Kicki, Sara, Henrik, Gunnarn, Charlotte, Anna, Johan, Linda, Christina, Gussing och Petra (ingen glömd hoppas jag). Nu kan jag äntligen siffervisan och det får man väl tacka Er för antar jag......

Min familj: Lars, Ulla och Chatrin för att ni alltid ställer upp och tror på mig – utan er hade jag inte varit där jag är idag.

Till sist vill jag tacka dig Maria, för att du är den du är. Stöttar mig när jag har det tungt och sprider glädje genom ditt smittsamma leende. Du är mitt ljus i mörkret!

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