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A Muscle Perspective on the Pathophysiology of Amyotrophic Lateral Sclerosis - Differences between extraocular and limb muscles

Vahid M. Harandi Umeå, 2016

Department of Integrative Medical Biology, Anatomy

Department of Clinical Sciences, Ophthalmology

a Umeå University SE-901 87 Umeå, Sweden

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Copyright © 2016 Vahid M. Harandi

Responsible publisher under Swedish law: The Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-445-5 ISSN: 0346-6612 New Series No: 1803 Electronic version available at http://umu.diva-portal.org Printed by: Print and Media, Umeå University Umeå, Sweden 2016 The original articles were reproduced with permission from the publishers. Figures 1, 2 and 3 were illustrated by Gustav Andersson.

Figures 5 and 6 were adapted from study I with the permission from PLoS One journal. Figures 7, 9 and 10 were adapted from study II. Figure 11 was adapted from study III with the permission from Investigative Ophthalmology & Visual Science journal. Cover illustration: Immunostaining of adult extraocular muscles; Wn5a (green), Neurofilament (red) and laminin (gray).

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“Be yourself; everyone else is already taken.”

(Oscar Wilde)

To my parents

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TABLE OF CONTENTS ABSTRACT ...................................................................................................................... VI ABBREVIATIONS ....................................................................................................... VIII LIST OF ORIGINAL PAPERS ..................................................................................... IX

1. INTRODUCTION ............................................................................. 1

1.1 Amyotrophic Lateral Sclerosis ............................................................... 2 Epidemiology .............................................................................................................. 3 Types of ALS: fALS and sALS .................................................................................. 3 SOD1G93A transgenic mouse - a mouce model for ALS research ............................... 4 Proposed pathogenesis of ALS ................................................................................... 4

1.2 Skeletal Muscles ....................................................................................... 7 Motor neurons ............................................................................................................. 7 Neuromuscular junction .............................................................................................. 7 Skeletal muscles – only a target in ALS? ................................................................... 8

1.3 Extraocular Muscles .............................................................................. 10 1.4 Neurotrophic Factors ............................................................................ 13

BDNF ........................................................................................................................ 14 NT-3 .......................................................................................................................... 14 NT-4 .......................................................................................................................... 14 GDNF ........................................................................................................................ 15 NGF .......................................................................................................................... 15 NTF receptors ........................................................................................................... 15 The importance of NTFs in motor neuron disease ................................................... 16 Changes in NTF receptors in different neurodegenerative diseases ......................... 17 Clinical trials of NTFs in ALS .................................................................... 18

1.5 Wnt Proteins .......................................................................................... 20 Wnt1 .......................................................................................................................... 21 Wnt3a ........................................................................................................................ 21 Wnt5a ........................................................................................................................ 22 Wnt7a ........................................................................................................................ 22 Wnt proteins in neurodegenerative diseases ............................................................. 23

AIMS ....................................................................................................... 24

2. MATERIAL AND METHODS ...................................................... 25

2.1 Ethical Considerations .......................................................................... 26 2.2 Muscle Sample Collection ..................................................................... 26

Mouse muscle sample (Study I, II and III) ............................................................... 26 Human muscle sample (Study III) ............................................................................ 27

2.3 Methods .................................................................................................. 28 RNA isolation and cDNA Synthesis ......................................................................... 28 Quantitative RT-PCR (qRT-PCR) ............................................................................ 28 Histochemistry (Study I) ........................................................................................... 29 Immunohistochemistry (Study II and III) ................................................................. 29

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2.4 Data Analysis (Study II and III) ........................................................... 31 Statistical analysis ..................................................................................................... 31

3. RESULTS ......................................................................................... 33

3.1 Morphological Alterations (Study I and II) ........................................ 34 3.2 mRNA Expression of NTFs (Study I) .................................................. 35

Control EOMs versus limb muscles .......................................................................... 35 Transgenic EOMs versus limb muscles .................................................................... 36 Control versus transgenic muscles ............................................................................ 36

3.3 Distribution of NTFs and their Receptors (Study II) ......................... 36 NTFs at NMJs ............................................................................................................ 37 NTFs at intramuscular nerve axons ........................................................................... 38 NTFs at muscle fibres ................................................................................................ 39 Distribution of NTF receptors at NMJs ..................................................................... 40

3.4 Distribution of Wnt Proteins in Muscles of ALS Donors and SOD1G93A Mice (Study III) .................................................................... 42

Intramuscular nerve axons ......................................................................................... 42 NMJs .......................................................................................................................... 43 Muscle fibres ............................................................................................................. 43

3.5 β-Catenin Expression in Muscle Fibres (Study III) ............................ 44

4. DISCUSSION ................................................................................... 47 4.1. Intrinsic Differences Between EOMs and Limb Muscles in Control Mice .......................................................................................... 48

Differences in NTFs between EOMs and limb muscles ........................................... 48 Differences in Wnts between EOMs and limb muscles ............................................ 49

4.2 Impact of Ageing versus ALS on NTFs and Wnts .............................. 49 4.3 Alterations of NTFs and their Receptors in ALS ............................... 50

Alterations of NTFs at mRNA level .......................................................................... 50 Alterations of NTFs detected by immunohistochemistry .......................................... 52 Decreased NTF receptors at NMJs ........................................................................... 53

4.4 Alterations of Wnt Proteins and β-catenin in ALS ............................. 54 Higher levels of Wnt1 and Wnt3a in nerve axons and muscle fibres of EOMs than of limb muscles .............................................................................................................. 54 Increased Wnt7a in muscle fibres in cytoplasmic region and subsarcolemma ......... 55 Wnt5a decreased at NMJs but unchanged in nerve axons and muscle fibres ........... 56 β-catenin .................................................................................................................... 56

CONCLUSIONS ............................................................................................................... 58 ACKNOWLEDGEMENTS ............................................................................................. 59 FUNDINGS ....................................................................................................................... 61 REFERENCES ................................................................................................................. 62

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ABSTRACT Background: Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder. ALS has been traditionally believed to be primarily a motor neuron disease. However, accumulating data indicate that loss of contact between the axons and the muscle fibres occurs early; long before the death of motor neurons and that muscle fibres may initiate motor neuron degeneration. Thus, the view of ALS is changing focus from motor neurons alone to also include the muscle fibres and the neuromuscular junctions (NMJs). While skeletal muscles are affected in ALS, oculomotor disturbances are not dominant features of this disease and extraocular muscles (EOMs) are far less affected than limb muscles. Why oculomotor neurons and EOMs are capable to be more resistant in the pathogenetic process of ALS is still unknown. The overall goal of this thesis is to explore the pathophysiology of ALS from a muscle perspective and in particular study the expression and distribution of key neurotrophic factors (NTFs) and Wnt proteins in EOMs and limb muscles from ALS donors and from SOD1G93A transgenic mice. Comparisons were made with age-matched controls to distinguish between changes related to ALS and to ageing. Results: Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4) were present in EOMs and limb muscles at both mRNA and protein levels in control mice. The mRNA levels of BDNF, NT-3 and NT-4 were significantly lower in EOMs than in limb muscles of early and/or late control mice, indicating an intrinsic difference in NTFs expression between EOMs and limb muscles. qRT-PCR analysis showed significantly upregulated mRNA levels of NT-3 and GDNF in EOMs but significantly downregulated mRNA levels of NT-4 in limb muscles from SOD1G93A transgenic mice at early stage. The NTFs were detected immunohistochemically in NMJs, nerve axons and muscle fibres. The expression of BDNF, GDNF and NT-4 on NMJs of limb muscles, but not of EOMs, was significantly decreased in terminal stage ALS animals as compared to the limb muscles of the age-matched controls. In contrast, NTFs expression in intramuscular nerve axons did not present significant changes in either muscle group of early or late ALS mice. NTFs, especially BDNF and NT-4 were upregulated in some small-sized muscle fibres in limb muscles of late stage ALS mice. All the four Wnt isoforms, Wnt1, Wnt3a, Wnt5a and Wnt7a were detected in most axon profiles in all human EOMs with ALS, whereas significantly fewer axon profiles were positive in the human limb muscles except for Wnt5a. Similar differential patterns were found in myofibres, except for Wnt7a, where its expression was elevated within sarcolemma of limb muscle fibres. β-catenin, a

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marker of the canonical Wnt pathway was activated in a subset of myofibres in the EOMs and limb muscle in all ALS patients. In the SOD1G93A mouse, all four Wnt isoforms were significantly decreased in the NMJs at the terminal stage compared to age matched controls. Conclusions: There were clear differences in NTF and Wnt expression patterns between EOM and limb muscle, suggesting that they may play a role in the distinct susceptibility of these two muscle groups to ALS. In particular, the early upregulation of GDNF and NT-3 in the EOMs might play a role in the preservation of the EOMs in ALS. Further studies are needed to determine whether these proteins and the pathways they control may be have a future potential as protecting agents for other muscles. Key words: Neuromuscular junctions, Extraocular muscles, Skeletal muscle, Neurotrophic factor, Wnt, Motor neuron disease, Amyotrophic lateral sclerosis, SOD1G93A mice.

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ABBREVIATIONS

ALS amyotrophic lateral sclerosis ANOVA analysis of variance AChR acetylcholine receptor BSA bovine serum albumin BDNF brain-derived neurotrophic factor cDNA complementary DNA CNS central nervous system EOM extraocular muscle fALS familial amyotrophic lateral sclerosis FITC fluorescein isothiocyanate FTD frontotemporal dementia Fz frizzled GDNF glial cell-derived neurotrophic factor H&E haematoxylin and eosin MIF multiply innervated muscle fibre mRNA messenger ribonucleic acid MyHC myosin heavy chain N-CAM neural cell adhesion molecule NF neurofilament NGF nerve growth factor NMJ neuromuscular junction nNOS neuronal nitric oxide synthase NT-3 neurotrophin-3 NT-4 neurotrophin-4/5 PBS phosphate buffered saline PCP planar cell polarity qRT-PCR quantitative reverse transcription polymerase chain reaction ROR2 receptor tyrosine kinase-like ophan receptor 2 sALS sporadic amyotrophic lateral sclerosis SIF singly innervated fibre SOD1 superoxide dismutase-1 TrkB tropomyosin-receptor kinase B TrkC tropomyosin-receptor kinase C WT wild type α-BTx α-bungarotoxin

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LIST OF ORIGINAL PAPERS

I. Harandi VM, Lindquist S, Kolan SS, Brannström T, Liu JX

Analysis of neurotrophic factors in limb and extraocular muscles of mouse model of amyotrophic lateral sclerosis. PLoS One. 2014; 9(10):e109833

II. Harandi VM, Gaied ARN, Brannström T, Pedrosa Domellöf F, Liu JX

Unchanged neurotrophic factors and their receptors correlate with sparing of extraocular muscles in Amyotrophic Lateral Sclerosis. (Submitted)

III. Harandi VM*, Mcloon LK*, Brannström T, Andersen PM, Liu JX

Wnt and extraocular muscle sparing in amyotrophic lateral sclerosis. Invest Ophthalmol Vis Sci (IOVS). 2014; 55(9): 5482- 5496

(* Joint first authors)

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Introduction

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1. INTRODUCTION “Do not wait to strike till the iron is hot; but make it hot by striking.”

(William Butler Yeats)

Introduction

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1. INTRODUCTION

1.1 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) was first discovered in 1869 by the French neuropathologist Jean-Martin Charcot (Charcot JM, 1874; Goldblatt D, 1968) and became well known when Lou Gehrig, the famous baseball player in the United States, ended his career after he was diagnosed with ALS in 1939. His illness brought national and international attention to the disease, thus, ALS is also known as Lou Gehrig’s disease. ALS is typically a late-onset, progressive neurodegenerative disease affecting both the upper motor neurons in the motor cortex and brainstem, and the lower motor neurons in the brain stem and spinal cord, leading to progressive muscle wasting, speech and swallowing difficulties, fasciculation, altered reflexes and spasticity (Figure 1). The death typically occurs eventually within 3 to 5 years of symptom onset, due to respiratory failure. Only about 25% of ALS patients survive for more than 5 years, and 10–20% of ALS patients have longer than 10 years survival (Chio A et al., 2008).

Figure 1: Schematic illustration of two motor neurons in the spinal cord and all the muscle fibres controlled by the motor neurons. Left side, large motor neuron and large muscle mass in healthy human being; Right side, small motor neuron and small muscle mass or athophied muscle fibres in ALS patient.

Introduction

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Epidemiology

ALS affects people all over the world in all ethnic groups. The incidence, prevalence, and mortality of the disease have slightly increased over the past 50 years (Worms PM, 2001). The worldwide incidence of the disease is approximately 2 to 3 new cases per 100,000 individuals per year (Logroscino G et al., 2008; Alonso A et al., 2009). In Sweden, the incidence has been shown to be 2.4 per 100,000 and the prevalence of the disease was reported to be 6-9 per 100,000 according to the report from Socialstyrelsen published in 2014. Some studies reported that men have as much as double the incidence of ALS in comparison to women, highlighting gender differences (Logroscino G et al., 2008). It became similar in both genders in later reports (Chio A et al., 2009; Fang F et al., 2009). This was speculated to be associated with smoking habits, occupation and better clinical evaluation in women in general (Logroscino G et al., 2008; Roman GC, 1996). A number of epidemiological studies have focused on environmental and exogenous risk factors for ALS such as exposure to pesticides, heavy metals, trace elements and chemicals, occupation, high levels of physical activity, smoking, male gender and low body mass (Bonvicini F et al., 2010; reviewed by Oskarsson B et al., 2015). Despite all efforts, the results have been inconclusive. Currently, the only established risk factors are ageing, male gender and family history (Ingre C et al., 2015). Types of ALS: fALS and sALS

Most ALS cases (90%) are sporadic (sALS) which occurr randomly without any obvious genetic component whereas approximately 10% of ALS cases are directly inherited and referred to as familial ALS (fALS) (Rowland LP and Schneider NA, 2001). A number of genetic loci and disease-causing mutations have been reported to be associated with ALS. The average age of onset for sALS is 55 to 65 years (Logroscino G et al., 2010; Orsini M et al., 2015) and 45 to 55 years for fALS patients (Orsini M et al., 2015). fALS can occur due to mutations in more than 20 genes (reviewed by Li HF and Wu Z, 2016), and the two most common genes are the gene encoding copper-zinc superoxide dismutase type 1 (SOD1) and the gene encoding C9orf72. SOD1 is a soluble protein, a ubiquitously expressed 153-amino acid long enzyme that converts superoxide into water and hydrogen peroxide. SOD1 was first discovered in 1993 (Rosen DR et al., 1993) and since then, 183 different mutations have been associated with ALS (http://alsod.iop.kcl.ac.uk). Eleven of these mutations have been expressed in transgenic rodent models giving rise to a rodent motor neuron disease phenotype rather similar to human ALS (reviewed by Andersen PM, 2006; reviewed by Andersen PM and Al-Chalabi A, 2011). In 2011, it was discovered that the most common genetic cause of both fALS (~ 40%) and

Introduction

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sALS (~ 7%) is the large intronic GGGGCC hexanucleotide repeat expansion in the C9orf72 gene (Dejesus-Hernandez M et al., 2011; Renton AE et al., 2011). Along with ALS, C9orf72 expansions are found in 25% of familial frontotemporal dementia (FTD) cases (Majounie E et al., 2012). In addition, TARDBP, FUS, OPTN, VCP, UBQLN2, and PFN1 are also the major genes underlying ALS. The genetic etiology of two-thirds of all fALS cases and approximately 10% of all sALS cases has not been identified yet (Renton AE et al., 2014). SOD1G93A transgenic mouse - a mouce model for ALS research

Over-expression of different variants of SOD1 has been among the most important tools in ALS research in the last decade. Although there are several other animal models expressing mutant SOD1, such as Drosophila melanogaster, Caenorhabadititis elegans, zebrafish and pigs, transgenic mouse models are the most practical animals to investigate the mechanisms in ALS. Mouse models of ALS have the advantage of close genetic and physiological similarities to human, being easy to be genetically manipulated, easy to be handled and their relatively high reproductivity rate over the other animal models to study the pathogenesis of ALS (Berthod F and Gros-Louis F, 2012). A number of transgenic mouse models over-expressing D90A, G127X, G85R and G93A have been generated to study ALS. However, the G93A mouse model has become by far, the most frequently used mouse model due to its conveniently short survival time and the predictable course of the disease (Gurney ME et al., 1994). SOD1G93A contains 23 to 25 copies of a flawed human SOD1 gene. This mouse model exhibits a disease that mimics an aggressive form of ALS, where animals exhibit muscle weakness and atrophy, and motor neuron loss at approximately 135 to 150 days of age. Proposed pathogenesis of ALS

The pathophysiology of ALS is still not fully understood and has been proposed to be related to a number of possible factors based on many studies (reviewed by Scarrott JM et al., 2015; reviewed by Mancuso R and Navarro X, 2015): a) Protein misfolding and aggregation: Protein misfolding and aggregation are closely associated with cell death and disease progression in various neurodegenerative diseases including ALS. SOD1 aggregation has been documented in transgenic mice (Bruijn LI et al., 1998; Jonsson PA et al., 2004 and 2006) and was extensively observed in fALS patients with SOD1 mutations (reviewed by Kato S et al., 2000; Jonsson PA et al., 2004; Stewart HG et al., 2006).

Introduction

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b) Defective axonal transport: Axonal transport is an important mechanism for cellular viability and transmission of nerve impulses. Morphological damage in axons of ALS patients (Hirano A et al., 1984a and b) and transgenic mice (Wong PC et al., 1995; Collard JF and Julien JP, 1995) clearly showed axonal transport deficit. c) Mitochondrial abnormalities: Involvement of mitochondria has been described in a number of neurodegenerative diseases, including ALS (reviewed by Lin MT and Beal MF, 2006). Mitochondrial damage was observed in the spinal cords of ALS cases and in most transgenic mouse models (Jaarsma D et al., 2000; Martin LJ et al., 2007; Bendotti C et al., 2001). d) Glutamate excitotoxicity: The glutamate level was shown to increase in the cerebrospinal fluid of ALS patients (reviewed by Rothstein JD, 1995). Riluzole, a glutamate antagonist is the only approved drug by “Food and Drug Administration” for the treatment of ALS. e) Oxidative stress: Redox homeostasis mediates cell stresses such as oxidative or nitrative stress, and its disturbance may lead to neurodegeneration and apoptosis (reviewed by Trachootham W et al., 2008; Lipton SA et al., 1993). Increased oxidative damage was observed in both sALS and fALS patients where the balance between the production of reactive oxygen species and the cell ability to detoxify malfunctions (Shaw PJ al., 1995; Ferrante RJ et al., 1997). f) Disturbance in RNA metabolism: It is the most recently discovered factor associated with the pathogenesis of the disease. TDP-43 protein has been shown to be a major component of the ubiquitin-positive neuronal inclusions and to play critical roles in the pathogenesis of ALS and FTD (Neumann M et al., 2006). TDP-43 discovery led to the identification of mutations in transactive response DNA binding protein, which encodes TDP-43 (Sreedharan J et al., 2008; Chio A et al., 2010). In addition, the discovery of mutations in Fused in Sarcoma (FUS) shortly after (Kwiatkowski TJ Jr et al., 2009; Vance C et al., 2009) and the C9orf72 gene, which also disrupt RNA metabolism (Dejesus-Hernandez M et al., 2011), indicates the importance of RNA metabolism in the pathogenesis of ALS. g) Deficit in growth factors: Previous studies have shown the critical roles of different neurotrophic factors in ALS (Anand P et al., 1995; reviewed by Elliott JL and Snider WD, 1996; Oppenheim RW, 1996). Deficit in growth factors causes motor neuron and cell death in ALS (reviewed by Redler RL and Dokholyan NV, 2012).

Introduction

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So far, the course of ALS is still not fully understood and there is no effective treatment to cure or at least significantly delay the progression of the disease. Currently, riluzole can only prolongs the life of ALS patients 2 to 3 months in most cases (Miller RG et al., 2007), especially when administrated at early stages of the disease (Andersen PM et al., 2007).

Introduction

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1.2 Skeletal Muscles

Motor neurons

There are approximately one hundred million muscle fibres in the human body, which are innervated by more than 120,000 motor neurons in the spinal cord (reviewed by Kanning KC et al., 2010). The upper motor neurons (corticospinal motor neurons) transmit motor information and signals along the axons to the brainstem and the spinal cord, where the cell bodies of lower motor neurons are located. Lower motor neurons receive these signals and transmit them to the specialized synapse formed between lower motor neurons and skeletal muscle fibres known as the neuromuscular junction (NMJ). Lower motor neurons trigger contraction of skeletal muscle and control fundamental body movements such as breathing and walking, which are controlled by spinal lower motor neurons, and swallowing and eye movements, which are controlled by cranial lower motor neurons. There are three types of lower motor neurons: alpha (α), beta (β) and gamma (γ) motor neurons. 1) α-motor neurons are the most abundant innervating extrafusal muscle fibres. 2) γ-motor neurons, innervating intrafusal muscle fibres in muscle spindles. 3) β-motor neurons, innervating both intra- and extrafusal muscle fibres. The α-motor neurons can be further classified into different subtypes according to the properties of target muscle fibres, such as fast-twitch fatigable, fast-twitch fatigue-resistant and slow-twitch fatigue resistant (Burke BR et al., 1973). Neuromuscular junction

The NMJ is structurally and functionally divided into three parts: presynaptic, synaptic and postsynaptic regions. NMJs comprise four major essential cell types, including nerve endings, muscle fibres, terminal Schwann cells and kranocytes (Sanes JR and Litchman JW, 1999; reviewed by Hughes BW et al., 2006; Court FA et al., 2008) (Figure 2). Motor neurons initiate action potentials and the nerve endings carry and transfer impulses from motor neurons to muscle fibres by releasing acetylcholine neurotransmitter that crosses the synaptic cleft and reaches the muscle fibre surface. Thereafter, acetylcholine binds to nicotinic acetylcholine receptors (AChRs) on the muscle fibre surface and ultimately induces contraction. Terminal Schwann cells are located at the motor endplate, in close proximity to the neuron-muscle synapse. Terminal Schwann cells act as a form of non-myelinating glial cells (reviewed by Griffin JW and Thomson WJ, 2008) and are involved in maintaining terminal axons and neuromuscular transmission (Jahromi BS et al., 1992; Reddy LV et al., 2003; Court FA et al., 2008). The nerve terminal is capped

Introduction

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by kranocytes, which are believed to be a fibroblast-like cell type (Court FA et al., 2008).

Figure 2: Schematic illustration of the four components comprising NMJ. Skeletal muscles – only a target in ALS?

ALS is traditionally hypothesized to be a pure motor neuron disease, which progresses from central nervous system (CNS) to perioheral nervous system (PNS) and subsequently affects muscles (Figure 1). However, this has been challenged by a number of studies (Gould TW et al., 2006; reviewed by Murray LM et al., 2010; Loeffler JP et al., 2016). Previous studies have shown that the accumulation of mutant SOD1 in postnatal motor neurons were not sufficient or critical to induce or accelerate disease progression in a mouse model of ALS, indicating the potential

Introduction

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involvement of non-neuronal cells in the pathogenesis of ALS (Lino MM et al., 2002; Pramatarova A et al., 2001). Previous studies have also shown that muscle-restricted SOD1 expression is sufficient to induce severe muscle atrophy, muscle strength reduction, sarcomere disorganisation and mitochondrial alterations (Dobrowolny G et al., 2008 and 2011). Selective skeletal muscle expression of hSOD1 is sufficient to cause motor neuron disease in mice, indicating that skeletal muscle may be an initiating cause for the pathological process in ALS (Wong M and Martin LJ, 2010). Another study has also shown the occurrence of muscle degeneration prior to any evidence of neurodegeneration or clinical symptoms in SOD1G93A mice (Marcuzzo S et al., 2011). Similarly, many studies have shown that retraction of nerve endings at NMJs occurs earlier than motor neuron loss (Frey D et al., 2000; Fischer LR et al., 2004; Dupuis L et al., 2009). In addition, early-impaired amplitude of both endplate potentials and miniature endplate potentials occur at early symptomatic stage (Rocha MC et al., 2013). A further spatiotemporal analysis of ALS progression in SOD1G93A mice has demonstrated that the actual denervation of NMJs occurs at 47 days followed by loss of motor axons and finally loss of α-motor neurons after 80 days. Data collected on a patient who died during the early stages of the disease also indicates that motor neuron denervation begins at the distal axon and proceeds retrogradely (Fischer LR et al., 2004). Similar findings have been also reported in other studies where NMJs in limb muscles were affected before lower motor neuron loss in the spinal cord (Dupuis L et al., 2009). Furthermore, early NMJ disturbances in ALS have been documented in other animal models such as dogs (Rich MM et al., 2002), zebrafish (Ramesh T et al., 2010), Caenorhabadititis elegans (Wang J et al., 2009), and Drosophila (Feiguin F et al., 2009). Taken together, these studies emphasize the active involvement of muscle cells in ALS and suggest that skeletal muscles may be actively involved in ALS, rather than a pure target in ALS. Therefore, skeletal muscle and NMJs need to be further studied in ALS.

Introduction

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1.3 Extraocular Muscles

There are six extraocular muscles (EOMs) in each orbit: four rectus (medial, lateral, superior and inferior) and two oblique (superior and inferior) muscles (Figure 3), which are the effector organ for the typical and diversified eye movements of mammals. In addition to these six EOMs, there is another muscle called retractor bulbi, which only exists in some species including mice, surrounding the optic nerve. However, the retractor bulbi is not a true EOM and has the properties that are similar to those of limb muscles.

Figure 3: Superior view of the EOMs showing the six EOMs and retractor bulbi. When EOMs contract, the eyeballs can produce highly specialised movements such as slow pursuit movements and rapid change from one point of fixation to another (saccades). The muscle fibres of the EOMs are generally organised into two layers: the orbital layer facing the orbital wall and the global layer facing the eye globe (Chiarandini DJ et al., 1979). The complexity of movements executed by the EOMs is reflected in their unique fibre type composition and structural organisation that

Introduction

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fundamentally differ from those of other skeletal muscles (Kjellgren D et al., 2004; Sadeh M, 2004; Spencer RF and Porter JD, 2006; McLoon Lk et al., 2011).

The EOMs are innervated by cranial nerves and have very small motor units (Fuchs AF et al., 1988) comprising approximately 10 muscle fibres per neuron (Barmack NH, 1977). The EOMs contain both singly innervated (SIFs) and multiply innervated muscle fibres (MIFs). They are unique in many aspects and therefore are considered a separate muscle class, allotype (Hoh JF and Hughes S, 1988). The unique properties of the EOMs are reflected in their expression profile that differs from that of limb muscle by over 300 genes and this difference has been verified at RNA, protein and metabolic levels by using a number of histochemical, cellular and molecular methods (Fischer MD et al., 2005). Skeletal muscles are generally divided into three major fibre types including; type I fibres (slow twitch, fatigue resistant), type IIa fibres (fast-twitch, fatigue resistant) and type IIB fibres (fast-twitch fatigable). This general classification does not apply to more specialised muscles (Stål P et al., 1994) and specially EOMs (Kjellgren D et al., 2003a and 2006). The EOMs fibre type composition is far more complex than that of skeletal muscles consisting of six fibre types in rodents including orbital MIF, orbital SIF, global MIF, global red SIF, global intermediate SIF and global pale SIF (Spencer RF and Porter JD, 1988). The muscle fibres in the EOMs do not fit this classification pattern and have a very complex MyHC composition, which may comprise up to five different MyHC isoforms in a single fibre (Kjellgren D et al., 2003a). Altogether, the muscle fibres in the human EOMs have a unique composition regarding the major contractile and relaxation proteins forming a continuum and reflecting the small size of the motor units and the finally tuned physiological properties of these muscles (Kjellgren D et al., 2003a, b and 2006). The EOMs are among the fastest muscles in the body (Chiarandini DJ et al., 1979) and are also extremely fatigue-resistant (Porter JD et al., 1996). In addition to these specific properties of EOMs, their high oxidative capacity and high fatigue resistance, which is uncommon among other skeletal muscles emphasizes on the complexity and uniqueness of EOMs (Bron AJ et al., 1997; Spencer RF and Porter JD, 1988). The most striking property of the EOMs is the unique behaviour of the muscles in different diseases. EOMs are vulnerable to some diseases, such as Grave’s disease, myasthenia gravis and Miller Fisher syndrome (Yanoff M and Duker J S, 1999; Mori M et al., 2001; Pedrosa Domellöf F, 2010 and 2012). However, unlike trunk and limb muscles, the EOMs are spared in many different muscular dystrophies including Duchenne and Becker muscular dystrophy, merosin deficient congenital muscular dystrophy, limb-girdle and congenital muscular dystrophies. These disorders severely affect the other muscles in the body and cause dramatic

Introduction

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weakness, loss of ambulation and early death (Pedrosa Domellöf F, 2012; Emery AEH, 2001; Sadeh M, 2004). In contrast to the devastating impact of ALS on the limb muscles of the same individulas, the EOMs are relatively spared in ALS, although ALS patients under long-term respiratory support do develop oculomotor disturbances, including ophthalmoparesis (Hayashi H et al., 1987; Palmowski A et al., 1995), indicating that the EOMs are affected at much later stage than the other muscles (Ahmadi M et al., 2010). After noticing the unique sparing of EOMs in ALS, our group has extensively studied the EOMs and limb muscles in ALS donors and in ALS transgenic mouse models in recent years. We observed that the morphological features of the EOMs were relatively well preserved compared to the limb muscles from the same ALS donors (Ahmadi M et al., 2010; Liu JX et al., 2011). Recently, we also observed that the EOMs, in contrast to limb muscles, retain a high degree of axonal presence at the NMJs in ALS patients (Liu JX et al., 2013) and in SOD1G93A transgenic mice (Tjust AE et al., 2012). These results indicate that the EOMs are capable of maintaining their NMJs at the late stage of the disease. Moreover, only mild changes in synaptic protein and laminin isoforms were observed in EOMs in comparison to the dramatic decrease in limb muscle of ALS patients (Liu JX et al., 2011). Differences between EOMs and limb muscles in ALS were also observed in SOD1G93A transgenic ALS mice where fragmentation of postsynaptic membrane, decreased density of AChRs and lack of nerve sprouting in denervated junctions were observed in limb muscles, but not in EOMs (Tjust AE et al., 2012; Valdez G et al., 2012). These results suggest that the EOMs are a promising model to better understand the pathophysiology of ALS and which may provide clues for future treatment approaches.

Introduction

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1.4 Neurotrophic Factors

Neurotrophic factors (NTFs) constitute a family of endogenous signalling proteins which may exert their trophic support through three prevalent modes and sites (reviewed by Ekestern E, 2004): paracrine (secretion from neighbouring cells), autocrine (motor neuron itself) or endocrine (target-derived delivery from distant cells). In paracrine signalling, NTFs can be secreted by other nearby cells including astrocytes, microglia and Schwann cells, so that the survival of developing neurons does not only depend on the supply of NTFs synthesized in their own targets but also the neighbouring cells (Askanas V, 1995). The autocrine production relies on motor neuron’s own ability to supply NTFs (reviewed by Ekestern E, 2004) whereas endocrine trophic support relies on delivery from distant cells, such as ependymal cells, blood or cerebrospinal fluid (Davies AM, 1996). NTFs were firstly described only as target-derived growth factors regulating the survival of neurons in the PNS. Skeletal muscles, in particular, were suggested to supply muscle-derived NTFs (Calof Al et al., 1984; Oppenheim RW et al., 1988 and 1993). Target-derived anterograde transport starts from upper motor neurons to lower motor neurons (Mufson EJ et al., 1999; Yan Q et al., 1992, 1993 and 1995) whereas retrograde transport initiates from muscle to the spinal cord and motor neurons meaning that target tissues secrete these growth factors that exert their effects retrogradely to the neuron’s cell body (Mufson EJ et al., 1999; Rind HB et al., 2002; Von Bartheld CS et al., 2001). NTFs have been suggested to function as target-derived molecules for motor neurons in the neuromuscular system (Yin QW et al., 1994; Bibel M and Barde YA, 2000; Dreyfus CF et al., 1999), where the NTFs act both pre- and postsynaptically and play a role in the functional maturation of synapses. In addition, NTFs also play critical roles in CNS with regulating synaptic plasticity, neuronal morphology and modulating axonal growth and neurotransmission (Chao MV, 2003; McAllister AK et al., 1999). NTFs are traditionally classified into three subgroups: 1) neurotrophic growth factor family including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and neurotrophin-6. The neurotrophic growth factors were the first to be characterized (Barde YA et al., 1982; Cohen S and Levi-Montalcini R, 1957; Levi-Montalcini R and Hamburger V, 1951; Maisonpierre PC et al., 1990); 2) glial cell-derived neurotrophic factor (GDNF), and 3) neuropoietic cytokines.

Introduction

14

BDNF

BDNF was isolated from pig brain for the first time (Barde Y et al., 1982; Leibrock J et al., 1989). BDNF can be produced in skeletal muscles (Matthews VB et al., 2009) and its expression is muscle activity dependent (Misgeld T et al., 2002). BDNF expression has been observed in skeletal muscles during motor neuron cell death (reviwed by Gould TW and Oppenheim RW, 2011). BDNF is an evolutionarily highly conserved protein (Tettamanti G et al., 2010) and presumably a prerequisite for more complex neuronal structures (Chao MV, 2000). Studies on BDNF have been shown its role in motor neuron survival and motor axon growth (Henderson CE et al., 1993; Yan Q et al., 1992; Sendtner M et al., 1992). Previous studies have shown that BDNF viral-mediated gene transfer in new-born mice provides significant neuroprotection against in vivo excitotoxicity (Bemelmans AP et al., 2006), and BDNF knockout mice appeared normal at birth, but the majority died within the first postnatal weeks (Klein R et al., 1993; Ernfors P et al., 1994a; Jones KR et al., 1994; Conover JC et al., 1995). NT-3

NT-3 can be synthesized in Schwann cells (Ernfors P et al., 1992) and its immunoreactivity is present in motor neurons in the cervical ventral spinal cord (Johnson RA et al., 2000) and skeletal muscle (Ernfors P et al., 1992; Henderson CE et al., 1993; Griesbeck O et al., 1995). The important role of NT-3 has been demonstrated in regulating the growth of muscle sensory neurons and maintaining proprioceptive sensory organs (Chen HH et al., 2002; Gorokhova S et al., 2009). NT-3 undergoes anterograde axonal transport to neurons and target tissue and functions as a neuromodulator (Altar CA and DiStefano PS, 1998). In addition, NT-3 can also be retrogradely transported from muscles to the cell body of motor neurons (Helgren ME et al., 1997). Thus, NT-3 affects both the central and the peripheral neurons during development and maturation (Maisonpierre PC et al., 1990). NT-3 also play critical roles in synaptic plasticity (Schnell L et al., 1994). NT-3 knockout mice develop movement deficits and die shortly after birth (Ernfors P et al., 1994b; Klein R et al., 1994), mainly due to neuronal death (Ernfors P et al., 1994b; Farinas I et al., 1994; Tessarollo L et al., 1997). NT-4

NT-4 was isolated for the first time from Xenopus laevis (Hallbook F et al., 1991). NT-4 is expressed by motor neurons in the spinal cord (Buck CR et al., 2000), and it plays an important role in maintenance of motor neurons (Stephens HE et al., 2005), growth and remodelling of adult motor neuron innervation (Beaumont E and

Introduction

15

Gardiner P, 2003). The production of NT-4 increases with muscle activity and correlates with neuromuscular growth (Funakoshi H et al., 1995). Therefore, NT-4 is important in maintaining NMJs and muscular performance. Muscle-derived NT-4 is required for maintenance of AChR and resistance to muscle fatigue (Belluardo et al., 2001). NT-4 knockout mice, unlike the knockout mice of other NTFs, remain viable and fertile with minimal neurological phenotypes (Conover JC et al., 1995; Liu X et al., 1995). GDNF

GDNF is a small extracellular peptide and was discovered in 1993 when the gene for GDNF was cloned (Lin LF et al., 1993). GDNF is widely expressed in the central and peripheral nervous systems (Schaar DG et al., 1993; Strömberg I et al., 1993; Golden JP et al., 1998), as well as in skeletal muscle (Trupp M et al., 1995; Suvanto P et al., 1996; Golden JP et al., 1999). GDNF is a very important factor in supporting motor neuron survival (Lin LF et al., 1993; Henderson CE et al., 1994), and also prevents programmed cell death of motor neurons during axotomy-induced degeneration of motor neurons (Yan Q et al., 1995). The prominent effect of GDNF on motor neurons and skeletal muscles is well established, and GDNF seems to be important in slowing degeneration in ALS (Henderson CE et al., 1994). Over-expression of GDNF in limb muscle correlates with hyper-innervation at NMJs in transgenic mice beyond the normal developmental period (Zwick M et al., 2001). NGF

NGF was the first NTF discovered being involved in the growth of sensory and sympathetic nerve cells (Levi-Montalcini R and Hamburger V, 1951; Cohen S, 1960). Since NGF’s function is limited to regulate the development and survival of motor neuromuscular system and differentiation of cholinergic neurons in CNS, and is generally not considered as a true motor neuron trophic factor (Seeburger JL et al., 1993; reviewed by Ekestern E, 2004). Therefore, NGF is not included in this thesis. NTF receptors

The NTFs exert their functions by binding to specific receptors (Barbacid M, 1994). BDNF, NT-3, and NT-4 bind to a common low-affinity receptor, p75NTR, but at different binding sites (Chao MV and Hempstead BL, 1995). BDNF, NT-3, and NT-4 bind to specific high-affinity tropomyosin tyrosine kinase (Trk) receptor: TrkB (specific for BDNF and NT-4) and TrkC (specific for NT-3).

Introduction

16

p75NTR belongs to the tumour necrosis factor receptor superfamily (Dechant G and Barde YA, 2002). p75NTR is expressed at very high level during development (Heuer JG et al., 1990), and it is present in various cell populations in CNS and PNS (reviewed by Roux PP and Baker PA, 2002). p75NTR plays a crucial role in both anterograde and/or retrograde axonal transportation of NTFs in sciatic nerve (Johnson EM et al., 1987). In the absence of Trk receptors, p75NTR binds and activates all the Trk-specific ligands with equal affinity. The major functions of p75NTR are to enhance or suppress Trk receptor activity, and to independently activate signalling cascades to induce apoptosis or survival (reviewed by Roux PP and Baker PA, 2002). Both BDNF and NT-4 have been shown to act on TrkB receptor in the pre- and postsynaptic regions of synapse (Fu AK et al., 1997; Wells DG et al., 1999). TrkB is expressed in spinal motor neurons and muscle fibres of rodents (Funakoshi H et al., 1993; Gonzalez M et al., 1999). TrkB has been shown to be a key factor in maintaining the properties of NMJs and neuromuscular transmission in adult mice (Mantilla CB et al., 2014). TrkC, a catalytic receptor, is abundantly expressed in both developing and adult nervous system (Lamballe F et al., 1994), specifically abundant in small γ-motor neurons in adult rats (Copray S and Kernell D, 2000). TrkC expression has also been shown in neuromuscular system. In addition, TrkC is expressed by mouse perisynaptic and myelinating Schwann cells at birth through adulthood and being unchanged after denervation. GDNF acts via a two-component receptor: GFRα-1 and protein tyrosine kinase (Ret) (Jing S et al., 1996; Treanor JJ et al., 1996; Worby JJ et al., 1996). The former interacts and associates primarily with GDNF (Airaksinen MS and Saarma M, 2002). GFRα-1 is expressed in high levels in developing and adult CNS and PNS (Widenfalk J et al., 1997). In addition, GFRα-1 is critical for motor neuron survival during development (Glazner JW et al., 1998). The importance of NTFs in motor neuron disease

BDNF has been shown to delay motor neuron degeneration and reduce muscle atrophy and motor axon loss in wobbler mutant mice, an ALS animal model (Mitsumoto H et al., 1994; Ikeda K et al., 1995). Other studies also showed that BDNF treatment slowed ALS progression, motor neuron degeneration and axonal loss using grip strength, rotarod test and muscle potentials in these mice (Ishiyama T et al., 2002 and 2004). In addition, exogenous BDNF were able to slow motor neuron degeneration in progressive motor neuropathy mice (Haase G et al., 1997; Sagot Y et al., 1998). NT-3 delivery via adenovirus-mediated gene can increase the life span by 50%, reduce motor axon loss and improve neuromuscular function in the mouse mutant of

Introduction

17

progressive motor neuronopathy (Haase G et al., 1997). NT-3 has been reported to be upregulated in limb muscle of ALS donors (Kust BM et al., 2002) but its immunoreactivity was reduced in the spinal cord in ALS donors, (Duberley RM et al., 1997). As NT-3 is known to promote motor neuronal survival and NT-3 knockout animals already have movement dysfunctions (Ernfors P et al., 1994b), the deficiency in NT-3 does not seem to be a cause of the disease (Duberlery RM et al., 1997). NT-4 has been shown to be beneficial in preventing spinal motor neuron death in neonatal rats and acts as a survival factor (Henderson CE et al., 1993). In addition, embryonic rat motor neurons have been shown to be mediated by NT-4 (Hughes RA et al., 1993; Wong V et al., 1993). NT-4 showed upregulation in muscle tissues in ALS and the upregulation increased along with the progression of the disease (Kust BM et al., 2002). The effects of GDNF on motor neuron survival, function, innervation and cell death have been investigated in a number of neurodegenerative diseases (Bohn MC, 2005). Decreased expression of GDNF in muscle and increased levels in the spinal cord has been reported in ALS patients when compared to controls post-mortem (Yamamoto M et al., 1996). Other studies in ALS muscles (biceps brachii, quadriceps, deltoideus, tibialis anterior and vastus lateralis) however, revealed increased GDNF mRNA levels in comparison to controls (Lie DC and Weis J, 1998; Yamamoto M et al., 1999; Grundström E et al., 1999). Exogenous GDNF delayed ALS onset, rescued motor neurons and increased lifespan of SOD1G93A mice (Li W et al., 2007; Acsadi G et al., 2002). GDNF over-expression in muscle cells but not in astrocytes extends the survival of ALS mice (Mohajeri MH et al., 1999). Intramuscular delivery of GDNF, rather than CNS-targeted delivery, was proposed to be a preferred therapeutic approach to achieve motor neuron protection in ALS (Li W et al., 2007; Suzuki M et al., 2008a). Changes in NTF receptors in different neurodegenerative diseases

It has been suggested that NTF receptors might be able to restore or improve CNS function after neuromuscular diseases (Pitts EV et al., 2006). P75NTR was upregulated in degenerating motor neurons in ALS patients (Kerkhoff H et al., 1991; Seeburger JL et al., 1993). Other studies also showed p75NTR receptor responses to pathological conditions by expressing in high degrees in ALS disease (Bussmann KA and Sofroniew MV, 1999; Scott AL and Ramer MS, 2010). Decreased BDNF and TrkB levels is involved in the pathogenesis of a number of neurodegenerative diseases including Parkinson’s disease, Alzheimer's disease, and Huntington's disease (Allen SJ et al., 2011; Lu B et al., 2013). Despite the upregulation of TrkB mRNA and protein levels in spinal cords of ALS donors (Seeburger JL et al., 1993; Mutoh T et al., 2000), dramatic decrease of tyrosine

Introduction

18

phosphorylation of TrkB receptor in spinal cords of ALS donors has been reported (Mutoh T et al., 2000). Studies on TrkC receptor in neurodegenerative diseases are rare. In a study on ALS patients, the mRNA level of TrkC was unchanged whereas the mRNA level of NT-3 was reduced in spinal motor neurons (Duberley RM et al., 1997). TrkC mRNA level was relatively unchanged after sciatic nerve axotomy but dramatically downregulated after sciatic nerve injury (Hammarberg H et al., 2000). GFR-α1 was reported to be significantly less abundant in motor neurons of the spinal cord of SOD1G93A mice (Zhang J and Haung EJ, 2006). In ALS patients muscle samples, the mRNA level of GFR- α1 were unchanged whereas GDNF was significantly changed (Yamamoto M et al., 1999; Mitsuma N et al., 1999). Clinical trials of NTFs in ALS

A number of clinical trials administering NTFs have been carried out in ALS patients in the past two decades (Henriques A et al., 2010; Tovar-y-Romo LB et al., 2014). In phase III clinical trial in ALS, treatment with subcutaneous recombinant BDNF failed to rescue motor neurons or slow down any disease progression (BDNF Study Group, 1999). Another clinical trail using subcutaneous and intrathecal delivery of BDNF had limited benefits and side effects including gastrointestinal symptoms and pain (Thoenen H and Sendtner M, 2002). Although BDNF is able to cross the blood-brain barrier by a saturable transport mechanism, significant penetration into the CNS is limited by the parenchyma, to which most systemically BDNF is bound (Pan Y et al., 1999). In Parkinson's disease, despite the successful results of intrastriatal infusions of recombinant GDNF in animal studies and Phase I clinical trials (Gould TW and Oppenheim W, 2011), a double-blind phase II trial for ALS disease failed (Lang AE et al., 2006). The failure of clinical trials of NTFs in ALS is probably due to (i) difficulties in succeeding with the appropriate delivery of sufficient quantities of NTFs; (ii) the multiple effects of NTFs on neuronal activity, which might lead to side effects including epileptic seizures in patients (Poo MM, 2001) or (iii) time of administration as trophic factor-based therapy could also be an obstacle if a short time frame of NTFs leads to motor neuron death (Tovar-y-Romo LB et al., 2014). Approaches to tackle these obstacles are considering the half-life of proteins, the dose and their ability to cross the blood-brain barrier by developing small molecules eventually able to activate the NTF receptors and stimulate NTF actions (Suzuki M and Svenden CN, 2008b; Tovar-y-Romo LB et al., 2014). Conventionally, ALS was believed to be a pure motor neuron disease. Thus, the target of NTFs delivery was the degenerating motor neurons through intrathecal delivery, subcutaneous administration, or inject into cerebrospinal fluid. In the past decade, more evidence showed that skeletal muscles play a critical role in ALS

Introduction

19

(Loeffler JP et al., 2016). Thus, the NTFs may need to be delivered directly to muscles. Importantly, the possible cause of clinical failure may be due to improper NTF (Ekestern E. 2004, Gould TW and Oppenheim RW, 2011). In previous clinical trials, BDNF has been mostly used. However, in many studies, BDNF failed to show rescue effects in the SOD1 ALS mouse model. Instead, GDNF seems to be a better candidate for therapeutic treatment of ALS (Li W et al., 2007, Acsadi G et al., 2002).

Introduction

20

1.5 Wnt Proteins

Wnt protein is another focus of the present thesis. Wnt proteins belong to a conserved family of secreted signalling molecules and play critical roles in a variety of biological processes including; embryonic development, cell proliferation, differentiation, migration and apoptosis. The Wnt signalling pathway is widely conserved throughout numerous species such as C.elegans, Drosophila, Xenopus and mammals. The Wnt pathway was originally discovered in Drosophila where the gene wingless (wg) demonstrated the ability to influence segment polarity during larval development (Nusslein-Volhard C and Wieschaus E, 1980). To date, 19 Wnt genes have been discovered in human and mouse genomes (Logan CY and Nusse R, 2004). Amomg the 19 identified Wnts, Wnt1, Wnt3a, Wnt5a and Wnt7a have been shown to be actively involved in regulating the pre- and postsynaptic structures of NMJs (Ataman B et al., 2008), AChR clustering (Cisternas P et al., 2014) and myogenic precursor cell proliferation (Otto A et al., 2008; reviewed by Budnik V and Salinas PC, 2011). Therefore, the four Wnt proteins were examined in the present thesis. Wnt proteins signal through three different pathways. One of the well-known pathways is called canonical pathway, which is β-catenin dependent (Clevers H, 2006). Increasing amount of evidence indicates the importance of Wnt/ β-catenin signalling in essentially all aspects of skeletal muscle development and maintenance (Church VL and Francis-West P, 2002). This pathway is a ligand dependent system where Wnt proteins bind to Frizzled (Fz) and a number of co-receptors leading to activation of dishevelled protein and ultimately increase of β-catenin in muscle fibres and nuclei (Reya T and Clevers H, 2005). β-catenin, the central player in the canonical pathway, is a cytoplasmic protein whose stability is dependent on destruction complex (reviewed by Clevers H, 2006). The other two pathways are non-canonical; β-catenin independent pathway: Wnt/Planar Cell Polarity (PCP) pathway and Wnt/Ca2+ pathway (Habas R and David IB, 2005; Clevers H, 2006). The non-canonical pathway controls a number of cellular processes including cell fate, cellular movement and myogenesis. The PCP pathway plays a role in patterning by instituting polarity of cells within a tissue, such as in the cochlea (Montcouquiol M and Kelley MW, 2003). In this pathway, Wnt signalling proteins proposed to be mediated through Fz co-receptors (He X et al., 2004), thereafter the signal is then transduced leading to activate dishevelled at the cell membrane. Wnt/Ca2+ pathway shares a number of components with the PCP pathway emerging from the stimulation of intracellular Ca2+ by Wnt proteins and Fz receptors (Kohn AD and Moon RT, 2005). However, this pathway found to be a critical signalling pathway for both canonical and the PCP pathway (reviewed by Komiya Y and Habas R, 2008). The two non-canonical Wnt signalling components, atypical protein kinase C and atypical receptor related tyrosine are crucial regulators

Introduction

21

of axon growth and plasticity during development and in adult nervous system (Keeble TR and Cooper HM, 2006; Wolf AM et al., 2008). The activity of Wnt proteins is influenced by their cellular context and their receptors (Grumolato L et al., 2010). Wnt proteins bind to a number of receptors including the amino-terminal cysteine-rich domain of the seven transmembrane Fz receptors (Bhanot P et al., 1996). Wnt proteins can also bind to tyrosine kinase activity receptors including atypical receptor related tyrosine kinase and orphan receptor tyrosine kinase (ROR2) (Inoue T et al., 2004; Logan CY and Nusse R, 2004). Wnt1

Wnt1 is highly expressed in nervous system, skeletal muscle fibres and at NMJs. Evidence for its role in nerve outgrowth and synaptic plasticity comes from several studies. Wnt1 is also involved in promoting nerve outgrowth (Hayashi Y et al., 2009). The development of dorsal interneuron was declined in Wnt1 knockout mice, suggesting that the lack of Wnt1 is defective in determination of dorsal interneuron (Muroyama Y et al., 2002). Wnt1 also fuctions to promote cell proliferation and over-expression of Wnt1 in thyrocytes stimulates cell growth (Kim WB et al., 2007). Wnt1 plays crucial roles for normal skeletal muscle development. C2C12 cells treated with Wnt1 shows higher levels of myogenic markers, such as MyoD (Huang J et al., 2015). Furthermore, Otto and his colleague revealed that exogenous Wnt1, together with Wnt3a and Wnt5a, could induce a great degree of satellite cell proliferation, and that Wnt1 accelerate the rate of cell diviosion (Otto A et al., 2008). The role of Wnt in formation of vertebrate NMJ during development was first discovered in Drosophila Wnt (Wingless) by Packard and his colleagues when they found that Wnt1 was secreted and clustered around synaptic botous at NMJs (Packard M et al., 2002). More importantly, they found that mutated Wnt1 resulted in a significant reduction in target-dependent synapse formation, which prevented NMJ expansion during muscle growth (Packard M et al., 2002). The authors suggested that Wnt1 plays a critical role in pre- and postsynaptic development although it has been shown that Wnt1 have no effect on AChR clustering (Luo ZG et al., 2002). Wnt3a

Wnt3a is a type of secreted protein and can initiate Wnt signalling in the Wnt/β-catenin signalling pathway (Chen Y et al., 2012a). Similar to Wnt1, Wnt3a also promote nerve outgrowth in canonical signalling pathway and co-expression of

Introduction

22

Wnt1 and Wnt3a has been observed during the development of dorsal neural tube (Ikeya M et al., 1997; David MD et al., 2010). Wnt3a also functions through its signalling pathway to regulate C2C12 myoblast differentiation (Huang J et al., 2015). C212 cells treated with Wnt3a shows increased level of myogenic markers including, MyoD (Huang J et al., 2015). Similar to Wnt1, Wnt3a has been shown to be required for satellite cell proliferation during adult skeletal muscle regeneration (Otto A et al., 2008; Tanaka S et al., 2011). Wnt3a role in synapse formation is also documented (Henriquez JP et al., 2008; Koles K and Budnik V, 2012). Wnt5a

Wnt5a encodes a cysteine-rich growth factor involving in several developmental processes in rodents. Wnt5a mediates growth factor-dependent, axonal branching and remodelling postsynaptic regions (Bodmer D et al., 2009; Li L et al., 2009; Cuitino L et al., 2010). Wnt5a also plays a role in specification and survival of motor neurons during development (Agalliu D et al., 2009). Similar to Wnt1 and Wnt3a, Wnt5a is also required for satellite cell proliferation during adult skeletal muscle regeneration (Otto A et al., 2008; Tanaka S et al., 2011). Wnt5a are highly expressed at NMJs and its role in remodelling postsynaptic regions (Korkut C and Budnik V, 2009; Cuitino L et al., 2010) and has been shown to induce postsynaptic assembly and function (Chen J et al., 2006). Wnt7a

Wnt7a is expressed in hippocampus during synaptogenesis and induces presynaptic differentiation by binding to its receptor Fz5 (Davis EK et al., 2008). In addition, Wnt7 can also signal directly to stimulate presynaptic assembly of synaptic vesicles and synaptic activity with coordinated clustering and apposition of synaptic proteins, by binding with dishevelled-1 (Ciani L et al., 2011). However, mice with Wnt7a-Desh-deficient may develop defects in spine morphogenesis (Ciani L et al., 2011). Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons, suggesting a potential role of Wnt7a in neurotransmitter release (Ahmad-Annuar A et al., 2006). By binding with Fz7, Wnt7a signalling has been shown to stimulate the growth and repair of skeletal muscles, by inducing the symmetric expansion of satellite stem cells through a non-canonical pathway (Bentzinger CF et al., 2014). It has been shown that over-expression of Wnt7a leads to myofibre hypertrophy and reduced myofibre damage in a mouse model of muscular dystrophy (von Maltzahn J et al., 2012a and b).

Introduction

23

Wnt proteins in neurodegenerative diseases

The activation of Wnt signalling pathway together with alterations of Wnts has been reported in many studies. Wnt5a and Wnt7a upregulation together with downregulation of Wnt4 are the consequence of muscle regeneration at the early stage of injury (von Maltzahn J et al., 2012c) whereas Wnt3a and Wnt7b are expressed at the end stage of injury (Polesskaya A et al., 2003; Brack AS et al., 2008). The upregulation of Wnt3a in the spinal cord dorsal horn after sciatic nerve injury has been reported (Itokazu T et al., 2014). Alterations in Wnt signalling have been implicated in several neuromuscular and neurological diseases including ALS, Alzheimer disease, Parkinson disease, frontotemporal dementia, muscular dystrophy, limb-gridle muscular dystrophy 2A and Huntington disease (reviewed by Nusse R, 2005). Studies on the spinal cord of adult mice have shown the expression of Wnt1, Wnt3a, Wnt5a and β-catenin by neurons and astrocytes as a result of the progression of ALS (Wang S et al., 2013; Chen Y et al., 2012a; Li X et al., 2013). Altered expression of Wnt1, Wnt2, Wnt4 and Wnt5a were found in the mouse model of ALS (Chen Y et al., 2012a; Wang S et al., 2013). Upregulation of Wnt3a was observed in the spinal cord of ALS mice both at the mRNA and protein levels (Chen Y et al., 2012a). In addition, Wnt1 protein was upregulated in the astrocytes in the spinal cords of ALS mice in comparison to control mice (Wang S et al., 2013). Similarly, the protein levels of Wnt4 were upregulated in the spinal cords of SOD1G93A ALS transgenic mice in comparison to control animals (Yu L et al., 2013). Wnt proteins have been shown to play important roles in NMJ maintenance and are able to activate signalling cascades in the pre- and postsynaptic compartments (reviewed by Budnik V and Salinas PC, 2011). As stated above, ALS has been traditionally believed to be a primary motor neuron disease progressing from the CNS to the PNS leading to muscle paralysis and death. However, evidence collected during the last decade shows that muscle denervation occurs long before the death of motor neurons being detected, suggesting that skeletal muscles may not be only a target of the disease but also play an active role in triggering motor neuron degeneration. In addition, while all other skeletal muscles are affected in ALS patients, the EOMs are far less affected than limb muscles. However, the underlying mechanisms for the selective sparing of EOMs are unknown. The present thesis evaluates the impact of ALS on EOMs versus limb muscles, focusing on the NTFs and Wnt proteins, in an attempt to understand the pathophysiology of ALS.

Aims

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AIMS

• To investigate and compare the mRNA levels of BDNF, NT-4, NT-3 and GDNF in EOMs and limb muscles at early and late stages in control and ALS transgenic mice.

• To compare the distribution of these NTFs and their receptors in EOMs and

limb muscles of control and terminal transgenic mice at early and late stages.

• To investigate Wnt1, Wnt3a, Wnt5a and Wnt7a in EOMs and limb muscles in control and ALS transgenic mice as well as in ALS donors and control subjects.

Material & Methods

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2. MATERIAL AND METHODS “There is not past, no future; everything flows in an eternal present.”

(James Joyce)

Material & Methods

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2. MATERIAL AND METHODS For standard techniques and experimental procedure, the reader is referred to a more detailed description given in the Material and Methods of study I, II and III.

2.1 Ethical Considerations

All animal studies were conducted in accordance with the European Communities’ Council Directive (86/609/EEC) and with approval from the Animal Ethical Committee of the Medical Faculty, Umeå University. All human muscle samples were collected from autopsy and used in accordance with the principles of the Declaration of Helsinki and with approval from the Research Ethical Committee of Umeå University and the Regional Ethical Review Board in Umeå, section for Medical Research.

2.2 Muscle Sample Collection Mouse muscle sample (Study I, II and III)

Transgenic SOD1G93A mice (Gurney ME et al., 1994) were originally obtained from Jackson Laboratories (Bar Harbour, Marine, USA) and were backcrossed with C57/BL6 Bom Tac for at least 20 generations. Muscle samples were taken from SOD1G93A mice at early age (42 to 52 days) and terminal stage (135 to 167 days) after the mice were euthanized with an intraperitoneal injection of pentobarbital. Terminal stage was defined as the stage when the animals were not able to stand on their legs or/and move around in cage and could not reach for food. Age-matched healthy littermates served as controls. Mice used in each study were divided into fur groups: early control, late control, early transgenic and terminal transgenic. Study I used 5 to 7 mice in each group. Study II used 4 to 7 mice in each group and study III used 3 to 4 mice in each group. Variation of the numbers of mice in each group was due to differences in numbers of mice for specimen of EOMs and limb muscles. The EOMs were dissected and collected from both orbits, and the limb muscle of both soleus muscle and gastrocnemius muscle were collected from both hind limbs. Figure 4 shows a terminal transgenic mouse and an age-matched control mouse (A) and their posterior limb muscle samples from the mice, respectively.

Material & Methods

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Figure 4: A) a terminal transgenic mouse (left) and a control mouse (right); B) limb muscle from terminal transgenic mouse (left) and from control mouse (right) of 150 days. Note the difference in body size and muscle size between transgenic mouse and control mouse.

Human muscle sample (Study III)

Human muscle samples of EOMs, biceps brachii, tibialis anterior, and vastus lateralis muscles were collected from autopsy of six ALS donors. Detailed information about the ALS donors was presented in study III. Human control muscle samples were collected from donors of autopsy with no known neuromuscular disease. The human controls were divided into “adult” and “elderly” groups. The “adult” was referred to an average age of 41 years for EOMs and 33 years for limb muscles, whereas the “elderly” were refered to an average age of 75 years for EOMs and 76 years for limb muscles. In total, there were four EOMs and five limb muscles in the adult group, and four EOMs and four limb muscles in the elderly group. For immunohistochemical analysis, the muscle samples were mounted on cardboard in embedding medium (OCT CRYOMOUNT, Histolab, Gothenburg, Sweden) and immediately frozen in propane chilled with liquid nitrogen, and stored at –80°C until further immunohistochemical analysis. For mRNA analyses, the muscle samples were excised and collected with additional care to avoid RNAase contamination and to free the muscle samples from any other unwanted tissues. Muscle samples were then submerged in RNAlater solution (Life Technologies Europe BV, Stockholm, Sweden) and incubated at +4°C overnight. Thereafter they were transferred and stored at -80°C until analysed.

Material & Methods

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2.3 Methods RNA isolation and cDNA Synthesis

Total RNA was extracted from muscle tissues using TRIzol Reagent according to the manufacturer’s protocol. In brief, 500µl and 1ml TRIzol (Invitrogen, Carlsbad, California, USA) for EOMs and limb muscles were added respectively based on their muscle tissue size. Samples were homogenized, centrifuged and suspended with chloroform, allowed to stand and centrifuged in order to obtain separating phase. The lower organic phase was precipitated with isopropanol, suspended, following by centrifugation. The RNA pellets were separated carefully and were rinsed with 1ml 75% ethanol followed by vortex and centrifugation. The remaining solution was discarded so that the RNA remained at the bottom of the tube. Nuclease-free water was added to the RNA pellets. RNA concentrations were determined using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity of RNA was assessed using an Agilent 2100 bioanalyzer and RNA 6000 Nano gel kit (Agilent Technologies, Waldbronn, Germany). The amount of RNA in the samples was calculated based on RNA concentration and integrity. Total RNA (1mg from limb muscles or 0.1mg from EOMs) was reverse transcribed using random hexamers and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). cDNA was stored at 20°C until further processing. Quantitative RT-PCR (qRT-PCR)

qRT-PCR was used to determine the relative mRNA levels in the examined muscle samples. qRT-PCR was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, CA, USA) using TaqMan Universal PCR Master Mix and the following TaqMan gene expression assays (Applied Biosystems) containing predesigned primers (Table 1). All reactions were carried out in 96-well optical reaction plates in triplicate according to the manufacture’s protocol (Applied Biosystems, California, USA). The thermal cycle run under the following conditions: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. To obtain appropriate standard curve for each gene, we tested and then used RNA from different tissues from which high levels of the interested gene was found. Spleen for NT3 and NT4, kidney for BDNF cerebellum for GDNF were used. Genes of interest were then normalized to the expression of β-actin using the Mouse ACTB (actin, beta) Endogenous Control Assay (NM_007393.1; Applied Biosystems).

Material & Methods

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For each reaction, the relative quantification was obtained as a ratio of the quantity (Qty) normalized by the standard curves to β-actin expression. The probe specifications that have been used in study I are listed in Table 1. Table 1: Primers and probes used in Study I

Probe name Code Manufacturer

BDNF Mm01334042_m1 Applied Biosystems NT-4/5 Mm01701591_m1 Applied Biosystems NT-3 Mm01182924_m1 Applied Biosystems

GDNF Mm00599849_m1 Applied Biosystems

Histochemistry (Study I)

Staining of hematoxylin and eosin was applied on cross-sections for visualizing general morphological features. After air-drying, slides were submerged in Harris haematoxylin solution (code: 01800; Histolab, Göteborg, Sweden) for 2 minutes. The slides were then immediately rinsed in distilled water for a few times, following with 0.1% acetic acid for a couple of seconds. After that, the slides were washed in lukewarm water for 4 to 5 minutes. Thereafter, staining with 1% Eosin in 70% ethanol was performed for 1 minute. Finally, the slides were dehydrated in absolute ethanol for 3 times, each for 2 minutes and then mounted in Pertex (code: 00840; Histolab). Immunohistochemistry (Study II and III)

Sections were brought to room temperature and air-dried for approximately 10 to 15 minutes. Either acetone or 2% paraformaldehyde in phosphate buffered saline (PBS) were used (fixation for staining of certain antibodies) to preserve tissue morphology and retain the antigenicity of the target molecules as an initial step in immunohistochemistry process. Sections were rinsed in 0.01M PBS, PH 7.2, containing 0.1% sodium azide and incubated for 15 minutes with appropriate normal serums of goat, donkey, or sheep from Jackson ImmunoResearch, West Grove, PA, USA. Afterwards, the sections were incubated with appropriate primary antibody (Table 2) at + 4°C overnight. All antibodies were diluted in 0.01M PBS containing 0.1% bovine serum albumin (BSA) and used at optimal dilutions.

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BSA was used in blocking unspecific bindings and in diluting solutions. As BSA contains IgG, which interferes with anti-goat and anti-sheep IgG secondary antibodies, PBS was used in those cases instead. After washing in PBS for 5 minutes 3 times, the sections were incubated again in normal serum and thereafter washed in PBS. The sections were then incubated with appropriate secondary antibody for 30 minutes at 37°C (Table 3). Control sections were treated the same as above, except that the primary antibody was omitted. No staining was observed in control sections. Alpha-bungarotoxin (α-BTX) was a ligend and used to visualize NMJs (study II and III). In double staining of α-BTX with oher anibodies, the secondary antibodies were just mixed together with α-BTX and applied to the sections. After incubation, the slides were washed with PBS and then mounted with Vectashield (Vector Laboratories; Burlingame, California, USA) and stored in 4°C until microscopy. Table 2: Primary antibodies used in Study II and III

Antigen Code Manufacturer Type * Dilution BDNF sc-546 Santa Cruz Biotechnology, Dallas, TX, USA Rabbit pAb 1:100

NT-4 sc-545 Santa Cruz Biotechnology, Dallas, TX, USA Rabbit pAb 1:100 NT-3 sc-547 Santa Cruz Biotechnology, Dallas, TX, USA Rabbit pAb 1:100

GDNF sc-328 Santa Cruz Biotechnology, Dallas, TX, USA Rabbit pAb 1:100 P75NTR G323A Promega, Madison, WI, USA Rabbit pAb 1:1000 TrkB ab18987 Abcam, Cambridge, UK Rabbit pAb 1:100 TrkC ab75174 Abcam, Cambridge, UK Rabbit pAb 1:100 GFR-α1 ab8026 Abcam, Cambridge, UK Rabbit pAb 1:100 MyHCI A4.951 DSHB, Iowa City, IA, USA Mouse mAb 1:300 MyHCI BAD5 DSHB, Iowa City, IA, USA Mouse mAb 1:50 MyHCIIa A4.74 DSHB, Iowa City, IA, USA Mouse mAb 1:300

Emb MyHC 2b6 DSHB, Iowa City, IA, USA Mouse mAb 1:50 NCAM AB5032 Millipore, Darmstadt, Germany Rabbit pAb 1:900 nNOS AB5380 Millipore, Darmstadt, Germany Rabbit pAb 1:5000

Wnt1 ab15251 Abcam, Cambridge, UK Rabbit pAb 1:500 Wnt3a ab28472 Abcam, Cambridge, UK Rabbit pAb 1:500 Wnt5a ab72583 Abcam, Cambridge, UK Rabbit pAb 1:500 Wnt7a ab100792 Abcam, Cambridge, UK Rabbit pAb 1:500

β-Cat E247 ab32572 Abcam, Cambridge, UK Rabbit mAb 1:300 NF MC726 DAKO, Glostrup, Denmark Mouse mAb 1:6000 Laminin PC128 The Binding Group, Birmingham, UK

Sheep pAb 1:30,000

Dystrophin GTX15277 GeneTex Inc., Irvine, CA, USA Mouse mAb 1:5000

* pAb = polyclonal antibody; mAb = monoclonal antibody

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In double staining of two different antibodies, the same staining procedures were repeated after the first staining. The slides were washed in PBS, added normal serum, and the second secondary antibody for incubation for 30 minutes at 37°C. The slides were finally washed and mounted with Vectashield (Vector Laboratories; Burlingame, California, USA) and stored at 4°C until microscopy.

Table 3: Secondary antibodies used in study II and III

Dye Host Reactivity

Code Source

Alexa 488 Donkey Rabbit 711-545-152 Jackson ImmunoResearch, West Grove, PA, USA Alexa 488 Goat Rabbit A-11034 Molecular Probes, Invitrogen Alexa 647 Donkey Sheep 713-605-174 Jackson ImmunoResearch, West Grove, PA, USA Alexa 568 Goat Mouse A-11031 Molecular Probes, Invitrogen

Rhodamine Redex Donkey Mouse 715-295-151 Jackson ImmunoResearch, West Grove, PA, USA

2.4 Data Analysis (Study II and III)

The sections were examined using a Nikon microscope (Eclipse, E800; Melville, NY, USA) equipped with a Spot RT colour camera (Diagnostic Instruments Inc., MI, USA). Computer generated images were processed using the Adobe Photoshop software (Adobe System Inc., Mountain View, CA, USA). EOMs and limb muscles were evaluated with respect to general morphology and the distribution of the NTFs. In cross sections of EOMs and limb muscle samples, the percentage of Wnt-positive nerve axons co-expressing NF was calculated out of the total number of axons positive only for NF. The percentage of myofibres positive for each of the Wnt isoforms out of the total number of myofibres in each cross section was also calculated. The percentage of NMJs co-expressing Wnts/NTFs and α-BTx out of the population of NMJs only positive for Wnt/NTF was calculated as well. Statistical analysis

Statistical analyses were conducted for all the measurements using unpaired t-test (StatView 4.5; Abacus Concepts, Inc., Berkeley California; study I and II). Statistical significance was determined by ANOVA and graphed by using Prism 6 software (GraphPad, San Diego, CA, USA; study III). All data were expressed as mean (M) and standard deviations (SD). Data was considered significant at P ≤ 0.05.

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3. RESULTS

”Ever tried. Ever failed. No matter. Try again. Fail again. Fail better.”

(Samuel Beckett)

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3. RESULTS

3.1 Morphological Alterations (Study I and II)

H&E staining revealed that in controls, the muscle fibres of EOMs were small and loosely arranged (Figure 5A) whereas the muscle fibres in limb muscles were large and tightly packed in a typical polygonal pattern (Figure 6A). No morphological alterations were seen in the EOMs of ALS transgenic mice at either early or terminal stages (Figure 5B).

Figure 5: H&E staining showing relatively preserved morphological architecture in EOMs terminal mice (B) as compared with EOMs from late control mice.

The morphology of the limb muscles in early ALS transgenic mice was similar to that of the controls. However, in the limb muscles of terminal stage mice, round atrophic and hypertrophic fibres with internal nuclei were encountered frequently (Figure 6B). In addition, fat replacement and areas with increased amounts of connective tissue were also observed.

Figure 6: H&E staining showing atrophic fibres and hypertrophic fibres with internal nuclei in terminal transgenic mice with arrows showing these morphological changes (B) as compared to late control mice (A).

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3.2 mRNA Expression of NTFs (Study I)

Comparison of mRNA levels of the four NTFs differed between EOMs and limb muscles, between controls and transgenic mice, and between early and late mice was shown in Figure 7.

Figure 7: mRNA levels of the four NTFs in EOMs and limb muscle from both control and ALS transgenic mice at early and late stages.

Control EOMs versus limb muscles

The mRNA levels of BDNF, NT-4 and NT-3 were significantly higher in limb muscles than in EOMs. BDNF mRNA level in limb muscles of both early and late controls were significantly higher than those in the EOMs of the corresponding stage. In early controls, NT-3 mRNA level was significantly higher in limb muscles than EOMs. NT-4 mRNA level was also significantly higher in limb muscles than EOMs from late controls.

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Transgenic EOMs versus limb muscles

Although BDNF mRNA expression level in the terminal stage was significantly higher in limb muscles than that in EOMs, this difference cannot be exclusively due to ALS since it was also present between the two muscles in late controls. GDNF and NT-3 mRNA levels were also higher in terminal limb muscles than in EOMs, although the difference was not statistically significant. Control versus transgenic muscles

In summary, in early transgenic mice, the mRNA levels of GDNF and NT-3 were significantly higher in EOMs whereas the mRNA level of NT-4 was significantly lower in limb muscles. The mRNA levels of all NTFs in the two muscles were higher in terminal transgenic mice than in controls, but only GDNF showed significantly higher mRNA level in transgenic mice than in controls in limb muscle.

3.3 Distribution of NTFs and their Receptors (Study II)

After examining the NTFs at mRNA level, we further investigated the distribution of the four NTFs in EOMs and limb muscles with immunohistochemistry to explore the alteration of NTFs at the structural level. NTFs were expressed in several structures including NMJs, nerve axons and muscle fibres (Figure 8).

Figure 8: BDNF was present in muscle fibres (A1 green), at NMJs (A2, red). NT-4 was present in nerve axons (B1 green, and colocalized with neurofilament (B2 red).

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NTFs at NMJs Control mice In both early and late control mice, the percentage of NMJs labelled with antibodies against the four NTFs was generally higher in limb muscles than that in EOMs (Figure 1A and 1B, study II). The difference of BDNF and NT-4 between the two muscles reached significant level in both early and late controls whilst the difference of NT-3 reached significant level in late mice. In brief, approximately half of the NMJs were positively stained for BDNF, NT-4, NT-3 and GDNF in early EOMs controls. There were no significant differences between early and late control EOMs with respect to all four NTFs. In limb muscles of both early and late control mice, the vast majority of NMJs were labelled with BDNF, NT-4 and NT-3 whereas fewer, but still the majority of NMJs was labelled with GDNF (Figure 2, study II). Transgenic mice The NMJs presented the most dramatic changes in pattern of immunostaining reactivity in transgenic mice. One of the major findings of this thesis was that BDNF, NT-3 and NT-4 was significantly decreased at NMJs in limb muscles at terminal transgenic mice as compared to late controls whereas no significant difference was found at NMJs in EOMs. In limb muscles of early transgenic mice, the percentage of NMJs labelled with the four NTFs showed no significant difference compared to early controls (Figure 2, study II). However, the percentage of limb muscle NMJs labelled with BDNF, NT-4 and GDNF was significantly decreased in terminal transgenic mice as compared to late controls (Figure 2, study II). Furthermore, the proportion of NMJs labelled with the four NTFs was significantly decreased in limb muscles along the time course of ALS. In contrast, there was no statistically significant difference in the percentage of labelled NMJs with all four NTFs between the EOMs control and transgenic mice, at both stages (Figure 2A and 2B, study II), although the number of labelled NMJs with GDNF was decreased in terminal transgenic mice as compared to early animals. Immunostaining intensity at NMJs In both early and late control mice, the immunostaining intensity was generally weak for BDNF and GDNF, moderate or strong for NT-3 and NT-4 in EOMs whereas it was generally weak for GDNF, moderate for BDNF and strong for NT-3 and NT-4 in limb muscles (Figure 3 and 4, study II). In early transgenic mice, the staining intensity of NMJs labelled with BDNF, NT-3, NT-4 and GDNF in limb muscles was similar to that of controls, whereas the immunoreactivity of NMJs labelled with the four NTFs became weaker in terminal transgenic mice. Interestingly, a number of NMJs in terminal limb muscles showed

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very strong labelling with BDNF (Figure 9 upper panel) and NT-4 (Figure 9 lower panel), showing higher staining intensity than those of controls. In EOMs of both early and terminal transgenic mice, the immunostaining intensities with the four NTFs generally resembled the patterns seen in age-matched controls (Figure 3, study II).

Figure 9: Immunolabelling showing BDNF (A1) and NT-4 (B1) exceptionally strong (green) at NMJs outlined by labelling of α-bungarotoxin, (red), in limb muscles of terminal transgenic mice.

NTFs at intramuscular nerve axons

Unlike the NTFs at NMJs in limb muscles, the immunoreactivity of the four NTFs in nerve axons did not change significantly in either early or terminal transgenic mice as compared to the respective controls. In both EOMs and limb muscles from early and late control mice, intramuscular nerve axons were strongly or moderately labelled with BDNF, NT-3 and NT-4, and weakly labelled with GDNF. The similar staining patterns were also observed in nerve axons in the two muscles from early and terminal transgenic mice (Figure 5 and 6, study II). The nerve axons showed co-expression of NTFs and NF in early and late controls and early transgenic nerve axons. In addition, it was noted that although the four NTFs were lacking in a variable number of nerve axons, the NTFs were present in the majority of intramuscular nerve axons in terminal limb muscles. However, NF was absent in more than half of the nerve axons in limb muscles of terminal transgenic mice (Figure 10).

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Figure 10: Immunostaining for NT-4 (green in A1 and B1) in nerve axons labelled by neurofilament (red in A2 and B2) in limb muscles of control mice (A1-A3) and of terminal transgenic mice (B1-B3). In the nerve axons of control mice, all nerve axons expressing NT-4 (A1) co-expressed neurofilament (A1-A3), whereas in terminal transgenic mice, the nerve axons labelling NT-4 (B1) either contained neurofilament (arrowheads) or not (arrows).

NTFs at muscle fibres

Muscle fibres in EOMs of both control and transgenic mice were weakly stained for the four NTFs in both early and late stages, although a small number of muscle fibres showed moderate staining with NT-3 and NT-4. No significant alterations in staining intensities with NTFs were observed in EOMs from transgenic mice. Similar staining patterns with the four NTFs were also found in limb muscle fibres from early and late controls as well as from early transgenic mice. In addition, in limb muscles from terminal transgenic mice, although the majority of muscle fibres showed similar staining pattern as late controls, a number of small-sized muscle fibres showed high immunostaining intensity with the four NTFs, especially with BDNF and/or NT-4. The fibres strongly labelled with BDNF were weakly labelled with NT-4 and vice versa. In addition, a small number of muscle fibres showed simultaneous increased immunoreactivity with BDNF and NT-4. The small muscle fibres with strong staining with antibodies against BDNF and/or NT-4 were generally unlabelled with antibody BA-D5 against slow MyHC, indicating that these fibres belonged to fast phenotype. To further define the properties of the muscle fibres showing strong reactivity for NTFs, several markers of muscle fibre denervation/regeneration (nNOS, N-CAM, MyHC embryonic) were used (Dubowitz V et al., 2013). In general in limb muscle fibres from early and late control mice, immunostaining with nNOS was found evenly distributed around the sarcolemma and concentrated at the NMJs. N-CAM was concentrated at NMJs and were detected with low amounts in the muscle fibres. In limb muscles of terminal transgenic mice, nNOS was absent from the sarcolemma from almost all the small-sized muscle fibres with strong labelling with

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antibodies against BDNF and/or NT-4. Notably, these muscle fibres could be categorised into three subgroups based on their staining patterns: (i) strong N-CAM but negative with embryonic MyHC; (ii) strong N-CAM and positive with embryonic MyHC; (iii) weak with N-CAM and strong with embryonic MyHC. Based on the assessment of nNOS, N-CAM and MyHC embryonic, the first two patterns might indicate that those small muscle fibres were probably denervated fibres and they displayed high levels of BDNF and/or NT-4. The third pattern may indicate that those muscle fibres were immature muscle fibres (Dubowitz V et al., 2013). Distribution of NTF receptors at NMJs

NTF receptors with low (p75NTR) and high (TrkB, TrkC and GFR-α1) affinity were examined at NMJs in EOMs and limb muscles from both control and transgenic animals. The vast majority of NMJs were generally labelled with all receptors in early and late control animals as well as early transgenic mice in both EOMs and limb muscles (Figure 8 and 9, study II). In terminal transgenic mice, the percentage of NMJs labelled with the four receptos was significantly decreased in limb muscles but not in EOMs (Table 4). Table 4: NMJs labelled with NTF receptors represented by mean±SD in late control and transgenic animals. Significant differences (p≤ 0.05) marked with a-i.

P75NTR There was no statistically significant difference in the percentage of NMJs labelled with anti-p75NTR between transgenic (95,5%±5) and control (95%±5,5) EOMs of terminal animals. In limb muscles however, the percentage of NMJs labelled with p75NTR was significantly declined in terminal mice (57%) when compared to late controls (97,5%) (Table 4(a)). There was also a significantly lower population of

Receptors Muscle Control Transgenic

P75NTR

EOMs 95%±5,5 (n=5) 95,5%±5 b (n=5)

Limb 97,5%±6 a (n=5) 57%±14,7 a, b (n=5)

TrkB

EOMs 96,2%±7,5 (n=4) 92,8%±6,8 d (n=5)

Limb 94%±5,2 c (n=6) 61%±4,8 c, d (n=5)

TrkC

EOMs 78,5%±6 f (n=4) 76,2%±9,7 g (n=4)

Limb 92,6%±8,8 e, f (n=5) 50%±8,9 e, g (n=5)

GFR-α1

EOMs 68%±12,5 i (n=4) 78,6%±9,3 (n=5)

Limb 97,5 %±5,6 h, I (n=5) 88%±3,6 h (n=5)

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NMJs labelled with p75NTR in limb muscles than EOMs in terminal transgenic mice (Table 4 (b)). TrkB In late control EOMs, 96,2% of NMJs were labelled with TrkB, which is similar to the EOMs in terminal transgenic animals in which 92,8% of NMJs were TrkB positive. The number of labelled NMJs significantly decreased in limb muscles of terminal transgenic from 94% to 61% (Table 4(c)). In addition, there was also a significantly lower population of NMJs labelled with TrkB in limb muscles than EOMs in terminal transgenic mice (Table 4(d)). The serial cross-sections of the terminal transgenic limb muscles showed that the majority of NMJs were either simultaneously labelled with TrkB and BDNF or with TrkB and NT-4 and the remaining were mostly labelled with neither TrkB nor BDNF/NT-4. In addition, a small number of NMJs were labelled with TrkB but unlabelled with NT-4. TrkC

The number of NMJs labelled with TrkC in EOMs at late control and transgenic stage were 78,5% and 76,2%, respectively. In terminal limb muscles, only 50% of NMJs were labelled with TrkC whereas the number of labelled NMJs was 92,6% in late controls, showing a statistically significant decline (Table 4(e)). There was a lower population of labelled NMJs with TrkC in EOMs compared to limb muscles in late controls (Table 4(f)). However, terminal transgenic mice had significantly lower population of NMJs labelled with TrkC in limb muscles compared to EOMs (Table 4(g)). In addition to the simultaneous presence and absence of NT-3 and TrkC at NMJs, the presence of NT-3 but absence of TrkC was also observed in a few numbers of NMJs in terminal limb muscles. GFR-α1

The number of NMJs labelled with GFR-α1 was significantly lower in late control EOMs than in late control limb muscles (Table 4(i)). However, this lower population of labelled NMJs in EOMs compared to limb muscles was not significant in terminal transgenic mice. The percentage of NMJs labelled with GFR-α1 showed no significant difference between EOMs in transgenic terminal mice (78,6%) as compared to controls (68%). In late control limb muscles, 97,5% of NMJs was labelled with GFR-α1, whereas 88% of NMJs were labelled with GFR-α1 showing significant decline in transgenic limb muscles (Table 4(h)). NMJs with presence of GFR-α1 but absence of GDNF were more frequently encountered than NMJs with simultaneous expression of GFR-α1 and GDNF due to the low proportion of NMJs labelled with GDNF.

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3.4 Distribution of Wnt Proteins in Muscles of ALS Donors and SOD1G93A Mice (Study III) In this study, the distribution of Wnt1, Wnt3a, Wnt5a, Wnt7a was investigated in intramuscular nerve axons, NMJs and muscle fibres from ALS donors and SOD1G93A mice. Intramuscular nerve axons EOMs In adult EOMs, 71% of nerve axons identified by neurofilament were labelled with the antibody against Wnt1 whereas the number of nerves axons positive for Wnt1 was significantly decreased to 42,5% in EOMs from ALS donors. Noticeably, the percentage of nerves axons in aged EOMs labelled with antibody against Wnt1 was only 11.6% (Figure 3A, study III). The percentage of axons positive for Wnt3a was very similar in adult EOMs (81%) and ALS EOMs (76%) whereas it was significantly decreased in aged EOMs (50%), indicating that the decline of Wnt3a expression was not due to ALS but rather to ageing (Figure 3B, study III). Unlike Wnt1 and Wnt3a, Wnt5a showed equally high populations of nerve axon profiles in all three examined EOM groups. Almost all the nerve axons were labelled with Wnt5a in EOMs from both adults and ALS donors whereas only 11% axons were unlabelled in EOMs from elderly (Figure 3C, study III). Wnt7a labelled the majority of axons in both adult (67%) and ALS (54,2%) EOMs, whereas it labelled significantly less axons in aged EOMs (7,9%) (Figure 3D, study III).

Limb muscles In limb muscles, the number of nerve axons labelled with Wnt1 was highest (31%) in adults, intermediate (23%) in ALS donors and lowest (16%) in elderly, but no statistically significant difference was found between any of the two groups (Figure 3A, study III). Similarly, the percentage of axons labelled with Wnt3a was not significantly different between any groups. There were 20,4% of the nerve axons labelled with Wnt3a in adult EOMs, 11,5% in elderly EOMs and 16,5% in ALS EOMs (Figure 3B, study III). Wnt5a labelled practically all nerve axon profiles in all examined limb muscles as it has been observed in EOMs (Figure 3C, study III).

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Approximately 50% of the axons were labelled with the antibody against Wnt7a in both adult and ALS limb muscles. However, the population of labelled axons was significantly higher in aged limb muscles compared to adult or ALS limb muscles (Figure 3D, study III). EOMs versus limb muscles A statistically significant difference in the percentage of Wnt1-positive nerve axons was observed between EOMs and limb muscles from adults and also between EOMs and limb muscles from ALS donors. The percentage was approximately 50% higher in EOMs than in limb muscles in both adult controls and ALS donors. Similarly, Wnt3a showed significantly higher percentage of labelled nerve axons in EOMs than in limb muscles in adults and ALS donors. In addition, the percentage of axons labelled with Wnt3a was also significantly higher in EOMs than limb muscles, in the elderly. In contrast, Wnt7a showed significantly lower percentage of labelled nerve axons in EOMs than in limb muscles in elderly subjects with 83% of nerve axons were labelled with Wnt7a in limb muscle but only 8% were labelled in the EOMs. NMJs Control and SOD1G93A mice All of the four examined Wnts were detected in the vast majority of NMJs in both EOMs and limb muscles from both early and late control mice. Similar to controls, in early transgenic mice, these Wnts were also present in the vast majority of NMJs in both EOMs and limb muscles. However, in terminal transgenic mice, the percentage of NMJs positive for the four Wnts was significantly decreased in limb muscle whereas it was maintained in the vast majority of NMJs in the EOMs. Muscle fibres EOMs The percentage of muscle fibres labelled with Wnt1 was significantly increased in EOMs from ALS donors as compared to adult EOMs, from 39% in adult EOMs to almost 100% in ALS EOMs. Less than one fifth of the muscle fibres expressed Wnt3a in adult EOMs and the percentage of muscle fibres expressing Wnt3a was significantly increased to 95% in EOMs from ALS donors. There was no significant difference in the percentage of muscle fibres labelled with Wnt5a between EOMs of adults and ALS donors, in which the vast majority of EOMs were labelled with

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Wnt5a. Less than one fifth of myofibres were labelled with Wnt7a in adult EOMs whereas slightly over half of the myofibres were labelled with Wnt7a. This increase was statistically significant between adults and ALS donors. Limb muscles The limb muscle fibres, in general, were not labelled with Wnt1 or Wnt5a in either adults or ALS donors whereas the percentage of muscle fibres stained with Wnt3a was significantly higher in healthy adult controls than in ALS donors. Wnt7a was present in approximately 50% of the myofibres in adult limb muscle. However, Wnt7a was detected in a higher percentage of myofibres in ALS donors although this increase was not statistically significant. Accumulation of Wnt7a in the subsarcolemma In control limb muscles from both human subjects and mice, Wnt7a was only present in a few myofibres around the fibre periphery. However, in limb muscles of ALS donors and terminal transgenic mice, bright rings of Wnt7a at the sarcolemma, either partially or entirely encircling the myofibre perimeter. Double staining with Wnt7a and dystrophin showed that Wnt7a was located subsarcolemmally (Figure 11).

Figure 11: Wnt7a (A), dystrophin (B) and merged (C) in terminal limb muscle.

3.5 β-Catenin Expression in Muscle Fibres (Study III)

β-catenin was present in the myofibres and in their myonuclei. Its expression showed different patterns and proportions in EOMs versus limb muscles, adults versus elderly, and human adults versus ALS donors. Labelling with β-catenin varied between global and orbital layers in EOMs from the three groups. The orbital layers were mostly unlabelled in sections treated with antibody against β-catenin whereas the global layers were mostly positive for β-catenin. Moreover, the number of β-catenin-positive fibres in the global layer was higher in elderly than in adults and ALS donors.

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In limb muscles, β-catenin was observed in approximately one third to half of the muscle fibres, in a mosaic pattern in both control and ALS donors. In limb muscles from elderly, a larger proportion of muscle fibres and nuclei were labelled with β-catenin.

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4. DISCUSSION

”Don’t judge each day by the harvest you reap but by the seeds that you plant.”

(Robert Luis Stevenson)

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4. DISCUSSION The present thesis primarily examined and compared the impact of ALS on NTFs and Wnts in EOMs and limb muscles in order to better understand the pathophysiology of ALS.

4.1. Intrinsic Differences Between EOMs and Limb Muscles in Control Mice

One important finding of the thesis is that there are intrinsic differences in the expression patterns of NTFs and Wnt proteins between EOMs and limb muscles, in control human subjects and animals. Differences in NTFs between EOMs and limb muscles

It is well established that EOMs differ from limb muscles in terms of fibre composition, innervation and structural organisation (Spencer RF and Porter JD, 2006), as well as gene profile in human (Fischer MD et al., 2005) and rat (Fischer MD et al., 2002). The present thesis, for the first time, showed that the expression of certain NTFs was significantly lower at the mRNA level (BDNF, NT-4 and NT-3) or at the protein level (BDNF, NT-4, NT-3, and GDNF) in EOMs than in limb muscles in early and/or late control mice. The reason of the intrinsic difference is unknown, but it may be related to differences in muscle innervation. Approximately, one third of muscle fibres in adult EOMs are multiple innervated (15% slow/slow tonic and 25% EOM fibres; Kjellgren D et al., 2003a; Liu et al unpublished data), whereas the muscle fibres in limb muscle convert from being transiently multi-innervated to single-innervated during development via axonal competition for innervation of a muscle fibre (Brown MC et al., 1976). The axons receiving muscle target-derived NTFs survive the process of synapse elimination whereas those lacking muscle target-derived NTFs are eliminated via programmed cell death (Oppenheim RW et al., 1990). Thus, NTFs seem to be less demanded in the EOMs than in the limb muscles in the perspective of controlling nerves’ surviving and maintenance.

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Differences in Wnts between EOMs and limb muscles

The present thesis also revealed significant differences in expression of Wnt proteins between the two types of muscles. The nerve axons presented significantly higher levels of Wnt1 and Wnt3a in EOMs than that in limb muscles and the EOM fibres contained higher Wnt1 and Wnt5a but lower Wnt3a and Wnt7a than limb muscle fibres. The underlying mechanisms for the intrinsic differences in Wnt expression between EOMs and limb muscles are currently unknown and beyond the scope of this thesis. The Wnt signalling pathway is involved in several processes including the development of muscle tissue and the formation of the motor neuron-muscle synapse (Cisternas P et al., 2014). Developmental origins of limb muscles and EOMs are distinct because limb muscles are generated from the somite-derived dermomyotome whereas EOMs originate from the cranial mesoderm (Sambasivan R et al., 2011; reviewed by Endo T, 2015). Alternatively, Wnt proteins might be released by different synaptic compartments meaning that Wnt signalling act via anterograde, retrograde or via autocrine signalling pathway through evolution which might explain the differences of the studied Wnts between EOMs and limb muscles (Ataman B et al., 2008). The expression of NTFs and Wnts in EOMs and limb muscles has not been previously investigated. The intrinsic differences in NTFs and Wnts expression reported here, may contribute to the sparseness of the EOMs in ALS.

4.2 Impact of Ageing versus ALS on NTFs and Wnts

Study II and III showed that NTFs and Wnts were significantly decreased at NMJs in limb muscles of terminal transgenic mice. The decline of the NTFs and Wnts cannot be attributed to ageing, as no significant differences in the expression of the NTFs and Wnts at NMJs of limb muscles were observed between early and late control mice. Therefore, the significant downregulation of the NTFs and Wnts in limb muscles of terminal transgenic mice was most likely due to the disease itself. Study III showed a dramatic decrease of Wnt1 in nerve axons in EOMs of ALS donors, as compared to adult controls. However, a significant decrease of Wnt1 was also found in nerve axons in EOMs from elderly, as compared to adult controls. The population of axons labelled with Wnt1 was almost triple in ALS donors than in elderly. Thus, the decrease of Wnt1 in the nerve axons in ALS donors is not due to ageing alone. In study III, significant decreases in populations of labelled nerve axons with Wnt3a and Wnt7a in EOMs were observed in elderly as compared to adults and ALS donors. However, no significant difference was observed between adults and

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ALS donors. The result suggests that the low level of Wnt3a and Wnt7a in nerve axons of ALS donors was due to ageing rather than the ALS disease.

4.3 Alterations of NTFs and their Receptors in ALS

The present thesis reported significant changes in NTF expression at both mRNA level and protein level, especially on the NMJs. Alterations of NTFs at the mRNA level Early upregulation of NT-3 and GDNF in EOMs in transgenic mice In the present thesis, NT-3 in EOMs was significantly higher in early transgenic mice than in early control mice. Alterations of NT-3 in muscle tissues and spinal motor neurons have been observed in ALS patients where NT-3 mRNA expression was upregulated in biceps brachii muscle (Kust BM et al., 2002) but decreased in spinal motor neurons (Duberley RM et al., 1997). As studies reported that NT-3 transfer retrogradely via axonal transport to spinal cord (Henderson CE et al., 1993; Yan Q et al., 1992; Braun S et al., 1996; Mitsumoto H and Tsuzaka K, 1999), it was believed that the motor neuron pathology in ALS triggered upregulation of NT-3 in muscles could transfer retrogradely via axonal transport to spinal cord to rescue the motor neurons (Duberley RM et al., 1997). Thus, the early upregulation of NT-3 in EOMs observed in the present thesis may be triggered by the early development of ALS to compromise the early impact of the disease on EOMs, leading to the relatively well-preserved EOM morphology. On the contrary, insufficient upregulation of NT-3, as observed in the limb muscles of the present thesis would fail to rescue the motor endplates and motor neurons, leading to motor endplate denervation. In study I, GDNF was also significantly upregulated in EOMs of early transgenic mice. Contrary to EOMs, in limb muscles of ALS mice, GDNF was not significantly upregulated at early but at terminal stage of ALS. In both transgenic mouse models and ALS patients, upregulation of GDNF expression has been reported in previous studies, which was suggested to be associated with motor neuron supporting and rescuing (Grundstrom E et al., 1999, 2000). Therefore, the early upregulation of GDNF in EOMs may be interpreted as an early response to ALS to protect the EOMs. The upregulation of GDNF in limb muscles of terminal transgenic mice might be interpreted as a secondary effect of motor neuron degeneration, as proposed by Kust (Kust BM et al., 2002). Taking the results of both EOMs and limb muscles into consideration, GDNF seems to play a role in protecting motor neurons from degeneration. The scenario is that the development

Discussion

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of ALS initiates an early upregulation of GDNF in EOMs, resulting in protection of the EOMs. However, ALS did not trigger an early upregulation of GDNF in limb muscles. The high level of GDNF in limb muscles of terminal transgenic mice may reflect an accumulation of GDNF due to the failure of retrograde transportation or may be associated with motor neuron rescuing, but in vain. Early downregulation of NT-4 in limb muscles in transgenic mice Muscle-derived NT-4 has been shown to be essential for maintenance of postsynaptic AChR regions and resistance to muscle fatigue (Belluardo N et al., 2001). Mice lacking NT-4 exhibit enlarged fragmented NMJs with disassembled postsynaptic AChR clusters, reduced AChR binding, and acetylcholinesterase activity (Belluardo N et al., 2001). Thus, NT-4 was proposed to be involved in growth and remodelling of adult motor neuron innervation by means of coordinated adaptation of neuromuscular performance to electronic stimulation and muscle exercise (Funakoshi H et al., 1995; Beaumont E and Gardiner P, 2003). Although NT-4 is not well studied in ALS, a study by in situ hybridization showed that the mRNA level of NT-4 in muscles of ALS donors is increased in the course of ALS progression and reached its peak at terminal stage (Kust BM et al., 2002). In the present study, NT-4 was significantly downregulated in limb muscles of early ALS mice compared to age-matched controls (Figure 4A, study I). Following the time course of ALS, NT-4 was, however, not affected further in limb muscles (Figure 4B, study I). On the contrary, NT-4 level was not significantly different in EOMs compared to controls at either stage (Figure 4C and 4D, study I), but it was indeed significantly upregulated following the time course of ALS (Figure 3B, study I). The present observation of early downregulation of NT-4 in limb muscles of ALS transgenic mice fits the early retraction of axons from NMJs (Frey D et al., 2000; Fischer LR et al., 2004), which lead to limb muscles de-innervation in terminal stage ALS mice. In contrast, the upregulation of NT-4 in the EOMs following the time course of ALS might be associated with maintenance of postsynaptic AChR regions or NMJs in EOMs. Unchanged BDNF The present study did not show significant changes in BDNF at the mRNA level in either EOMs or limb muscles between the control and the transgenic mice. The results are consistent with a previous study where the mRNA level of BDNF in muscles from ALS donors was in the same range as that of healthy controls (Grunstrom E et al., 1999). However, in different studies on skeletal muscle of mammals, the levels of BDNF at both mRNA and protein levels have varied from no expression (Schecterson LC and Bothwell M, 1992; Sakuma K et al., 2011), to low (Henderson CE et al., 1993; Gomez Pinilla F et al., 2001) to very high

Discussion

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expression (Griesbeck O et al., 1995), indicating either complexity of BDNF expression in skeletal muscles or technical difficulties in measuring it. The expression of BDNF was upregulated after muscle denervation in neonatal rats suggesting a role in rescuing motor neurons from degeneration (Koliatsos VE et al., 1993). Similarly, BDNF mRNA and protein levels were increased in limb muscles of ALS patients in early stage of the disease (Kust BM et al., 2002) suggesting that degenerating motor neurons are exposed to elevated levels of muscle-derived BDNF. Alterations of NTFs detected by immunohistochemistry Decreased NTFs at NMJs in limb muscles of terminal transgenic mice The percentage of NMJs labelled with BDNF, GDNF and NT-4 showed a significant decrease in limb muscles between terminal transgenic mice and the age-matched controls. However, there was no significant decrease in the number of labelled NMJs in EOMs between the terminal transgenic mice and late controls. Furthermore, the percentage of NMJs labelled with the four NTFs was significantly decreased in limb muscles of terminal transgenic mice as compared to early transgenic mice, suggesting that the percentage of labelled NMJs was declined significantly along the time course of the disease. Previous studies have shown that NTFs play crucial roles in regulating synapse development, growth and plasticity. While BDNF was able to trigger synaptic potentiation, growth and repair (Je HS et al., 2013; Lu B et al., 2013), GDNF was associated with hyperinnervation of NMJs and multiple end-plate formation (Nguyen QT et al. 1998; Zwick M et al., 2001), and injection of exogenous BDNF and NT-4 led to enhanced neuromuscular transmission in adult rat (Mantilla CB et al., 2004). Interestingly, in terminal stage ALS mice of the same model as the present study, we have previously observed disconnection between muscle fibres and motor nerve endings at NMJs in limb muscles, but not in EOMs (Tjust AE et al, 2012). Similar findings (denervation on NMJs) have also been reported in other studies (Fischer LR et al., 2004; Frey D et al., 2000; Dupuis L et al., 2009). Nevertheless, it is still unknown whether the NTFs’ downregulation on NMJs in limb muscles of late stage ALS mice was related to the initiation or a consequence of NMJs denervation in ALS. Unchanged NTFs presence in intramuscular nerve axons The intramuscular nerve axons in either early or terminal transgenic mice did not present significant changes in NTFs expression. However, NF, a major component of neuronal cytoskeleton in nerve axons was absent in more than half of the nerve axons in limb muscles of terminal transgenic mice. Lack of NF has previously been

Discussion

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observed in nerve axons of skeletal muscles (Liu JX et al., 2013) and in motor neurons of spinal cord (Bergeron C et al., 1994; Menzies FM et al., 2002) in ALS patients, and in cell culture of ALS models (Menzies FM et al., 2002). Whether the strong expression of NTFs is associated with the diminishing of NF is presently unknown, and was beyond the focus of the present thesis. Increased BDNF and NT-4 in small-sized limb muscle fibres In muscle fibres of either EOMs or limb muscles, expression of NTFs generally did not present significant difference between control and transgenic mice at either early or late stage. However, in some small-sized muscle fibres from limb muscles of terminal transgenic mice, the expression of BDNF and NT-4 was significantly increased and co-expression of the two NTFs was seen in some of the muscle fibres. In addition, most of the fibres were positive for N-CAM but negative for nNOS. N-CAM is normally expressed at NMJs in healthy muscles and becomes concentrated in sarcoplasmic region in denervated and regenerated fibres (Covault J et al., 1987; Sanes JR et al., 1986; Dubowitz V et al., 2013), whereas nNOS is normally expressed in the sarcolemma of healthy muscles and absent from denervated muscle fibres (Dubowitz V et al., 2013). As these fibres were either positive to embryonic MyHC or negative to embryonic MyHC, we postulated that the small-sized fibres with upregulation of BDNF and NT-4 were either denervated or regenerated. The results of NTFs obtained with qRT-PCR at mRNA level in study I and with immunohistochemistry at protein level in study II were not fully comparable. The possible reasons are: (i) the mRNA expression detected with qRT-PCR is a sum-up of all structures within the muscle tissue whereas the present study was a visualization of the NTFs in individual structures; (ii) NTF alterations in each individual structure were highly different; (iii) the mRNA level may not reflect the rate of protein synthesization and the steady state of protein content (Vogel C and Marcotte EM, 2012). Decreased NTF receptors at NMJs

Study II revealed a significant decrease of the four NTF receptors, p75NTR, TrkB, TrkC and GFR-α1 at NMJs in limb muscles of terminal transgenic mice. Such significant decline was not found at NMJs in EOMs of terminal transgenic mice. Decreased NTF receptors have been previously reported in other studies on SOD1 transgenic mice and ALS patients. TrkB showed dramatic less phosphorylated in spinal cords of ALS donors (Mutoh T et al., 2000) and GFR-α1 was decreased significantly in motor neurons of the spinal cord in SOD1G93A mice (Zhang J and Haung EJ, 2006). However, upregulated mRNA levels of P75NTR have been reported in degenerating motor neurons in ALS patients and terminal stage SOD1G93A mice (Kerkhoff H et al., 1991; Seeburger JL et al., 1993; Lowry KS et al., 2001). In

Discussion

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addition, unchanged mRNA level of TrkC was observed in spinal motor neurons in ALS patients (Duberley RM et al., 1997). The controversial results in the studies can be explained partly by the fact that NTFs were examined at mRNA versus protein levels. Study II also showed that NTFs and their specific receptors were decreased simultaneously at NMJs in limb muscles of terminal transgenic mice. However, such simultaneous decreases were not reported in other studies. The mRNA level of NT-3 was decreased in spinal motor neurons in ALS patients and the mRNA level of TrkC was unaltered (Duberley RM et al., 1997). In addition, while the mRNA level of GDNF was significantly reduced in muscle specimen of ALS patients, the GFR- α1 mRNA level was unchanged (Yamamoto M et al., 1999; Mitsuma N et al., 1999). It is unknown whether the simultaneous decreases in NTFs and the receptors observed in the study are just coincident or whether there is a causal relationship between them. Nevertheless, a previous study of BDNF/TrkB signalling in ageing revealed that while endogenous BDNF was less available at NMJs of diaphragm at 18 months of age, reduction in TrkB occurred at 24 months of age, indicating that changes in NTFs and the receptors have a sequential relationship (Greising SM et al., 2015).

4.4 Alterations of Wnt Proteins and β-catenin in ALS

During the last decade, extensive research has shown abnormal Wnt signalling in different neurodegenerative diseases (Caricasole et al., 2004; De la cruz e silva OA et al., 2010; Godin JA et al., 2010). In ALS however, the relationship between Wnt proteins/Wnt signalling pathways and the pathogenesis of the disease is still not fully understood. In ALS transgenic (SOD1G93A) mice, alteration of a number of Wnt proteins including Wnt2, Wnt3a, Wnt7a and Wnt4 together with β-catenin was observed in spinal cords at both mRNA and protein levels (Chen Y et al., 2012a, b). In addition, expressions of Wnt receptors were also found to be altered in spinal cord of ALS transgenic (SOD1G93A) mice (Yu L et al., 2013). The present thesis reported significant alterations in expression of certain Wnt proteins in different structures of ALS transgenic mice and of ALS patients. Higher levels of Wnt1 and Wnt3a in nerve axons and muscle fibres of EOMs than of limb muscles In ALS donors, significantly higher levels of Wnt1 and Wnt3a were observed in nerve axons of EOMs than in limb muscles. In adult controls, similar trends of higher levels of Wnt1 and Wnt3a in nerve axons of EOMs than of limb muscles were also observed. In elderly controls however, the level of Wnt1 in nerve axons of EOMs was similar to that of limb muscles, whereas the level of Wnt3a in nerve

Discussion

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axons of EOMs was significantly higher than that of limb muscles. On basis of the results, we suggest that 1) there are intrinsic higher levels of Wnt1 and Wnt3a in nerve axons of EOMs than of limb muscles; 2) the higher levels of Wnt1 in EOMs than in limb muscles of ALS donors was not due to ageing alone. Previous studies have shown that Wnt1 acts in both the peripheral and the central nervous systems at pre- and post-synaptical regions to control cytoskeletal dynamics in the nerves as well as assembly and clustering of the postsynaptic apparatus (Ataman B et al., 2008; Salinas PC and Zou Y, 2008), and that Wnt3a is involved in promoting nerve outgrowth (Endo Y et al., 2008; David MD et al., 2010). Wnt1 has also been shown to prevent neurite elimination (Hayashi Y et al., 2009), and early deletion of Wnt1 at the embryo stage resulted in the absence of cranial nerves III and IV, and disruption of the aneural EOMs (Mastick GS et al., 1996; Porter JD and Baker RS, 1997). Therefore, we suggest that in human beings, the intrinsic higher levels of Wnt1 and Wnt3a in nerve axons of EOMs than of limb muscles might function to protect motor neuron innervation of the muscles with less disturbance of ocular motility. In ALS donors, both Wnt1 and Wnt3a were significantly increased in muscle fibres of EOMs, whereas Wnt1 was unchanged and Wnt3a was significantly decreased in limb muscles in comparison to adult controls. Wnt1 has been shown to help regulating muscle specification and NMJ formation in development, and play a critical role in synaptic plasticity and muscle regeneration in mature animals (Stersn MH et al., 1995; Packard M et al., 2002). Wnt3a was also suggested to play an important modulatory role in the formation of NMJs, including number and size (Henriquez JP et al., 2008). The present observation of the significant increase in Wnt1 and Wnt3a in muscle fibres of EOMs of ALS donors was thus suggested to be associated with the selective sparing of EOMs. Taken together, the increased expression of Wnt1 and Wnt3 in nerve axons and muscle fibres of EOMs in ALS donors suggested a potential link between the two Wnt proteins and the sparing of the ocular motor neurons and the EOMs in ALS. This hypothesis is supported by our early observation of intact innervation of NMJs in EOMs in comparison of retraction of nerve axons from NMJs in limb muscles of ALS (Tjust AE et al., 2012; Liu JX et al., 2013).

Increased Wnt7a in muscle fibres in cytoplasmic region and subsarcolemma The most important finding with Wnt7a in Study III was that Wnt7a increased significantly in muscle fibres of EOMs in ALS donors as compared to adult controls. Wnt7a was also increased in limb muscles in ALS donors, but the increase did not reach the significant level. However, there was a specific feature for Wnt7a that it was expressed at the periphery of a large number of myofibres, especially in

Discussion

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small-sized fibres of limb muscles in both ALS donors and terminal ALS transgenic mice. It has been reported that when dystrophic muscle fibres fused to Wnt7a-loaded satellite cells, the muscle fibres presented increased growth and strength via an activation of Wnt signalling pathway by binding to its receptors (Bentzinger CF et al., 2014). The authors suggested that Wnt7a could be released from intracellular storage to induce the signalling events leading to the fibre growth response (Bentzinger CF et al., 2014). In the present study, Wnt7a accumulation inside the sarcolemma of muscle fibres in ALS limb muscles was observed. We supposed that muscle fibre-secreted Wnt7a might be blocked in Wntless/Evi (the Wnt secretory pathway) as evidenced by the Wnt7a accumulation inside the sarcolemma, leading to terminal stage of muscle dysfunction in ALS. Wnt5a decreased at NMJs but unchanged in nerve axons and muscle fibres Wnt5a showed constantly high levels in nerve axons and muscles fibres of both EOMs and limb muscles in both control subjects and ALS donors. In addition, comparison between Wnt5a levels in different groups did not present any significant difference. The population of NMJs labelled for Wnt5a and the other three Wnt proteins Wnt1, Wnt3a and Wnt7a, showed a significant reduction in limb muscles of terminal ALS transgenic mice, but not in EOMs. The present Wnt results, together with the results in Study II showing significant decrease in NTFs at NMJs, correlated with the previous findings that NMJs were denervated in the limb muscles of the ALS mouse model, only at the terminal stage but not at the early stage (Tjust AE et al., 2012). Although Wnt proteins play a key role in NMJ development, maintenance and remodelling (Koles K and Budnik V, 2012; Wu H et al., 2010), it is unknown whether the decreased Wnt at NMJs in limb muscles of terminal stage ALS mice was the initiation or the consequence of NMJs denervation in ALS. β-catenin

Our results showed that β-catenin expression was present in a subset of myofibres in both EOMs and limb muscles. Although the distribution of β-catenin expression differed in EOMs, no apparent correlation between β-catenin-positive myofibres with any of the four Wnt isoforms in EOMs or limb muscles was found. However, it has been suggested that β-catenin positive myofibres use the canonical Wnt signalling pathway (Speese SD and Budnik V, 2007). Complementary experiments could clarify whether the β-catenin-positive myofibres are involved in the canonical

Discussion

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Wnt signalling pathway and reveal the functional consequences of the upregulated β-catenin, as it has been associated with many processes in muscle, including the regulation of AChR clustering, presynaptic function (Farias GG et al., 2010) and axonal remodelling (Hall AC et al., 2000). In addition to the role of canonical pathway in ALS pathogenesis, the alterations of non-canonical Wnt signalling components have been also shown to contribute to neurodegeneration in ALS where atypical Protein kinase C and Wnt receptor, atypical receptor related tyrosine kinase were upregulated in the spinal cords of SOD1G93A mice (Tury A et al., 2014), suggesting that both canonical and non-canonical Wnt signalling pathways might contribute to the pathogenesis of ALS.

Conclusions

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CONCLUSIONS • There are intrinstic differences between the EOMs and the limb muscles

regarding patterns of expression of BDNF, NT-3, NT-4 and GDNF, as well as Wnt1, Wnt3a, Wnt5a and Wnt7a.

• The expression patterns of Wnt1, Wnt3a and Wnt7a in the nerve axons of

the EOMs significantly decreased with age.

• The intrinsic differences between EOMs and limb muscles above together

with the early upegulation of GDNF and NT-3 mRNA in EOMs and downregulation of NT-4 mRNA in limb muscles, as well as the increased levels of Wnt1, Wnt3a and Wnt7a in nerve axons and muscle fibres in EOMs might be instrumental for the distinct involvement of these muscle groups in ALS.

Acknowledgements

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ACKNOWLEDGEMENTS

I would like to express my great debt of gratitude towards all the people who have supported, helped and guided me through the years I was working in the IMB Department. This thesis would not have been possible without their ability to show me the light at the end of tunnel. It has been an absolute privilege to work with them. I would specifically like to thank the following people: Special mention goes to Associate Prof. Jingxia Liu, my supervisor, for all your support and encouragement through my PhD. For always having your door open and being caring. Your advices were always helpful. I genuinely appreciate all your contributions of time and patience. Prof. Fatima Pedrosa-Domellöf, my assistant supervisor, for your generosity, enthusiasm and guidance throughout all these years. I have certainly learned very much from you in science but also in other aspects of life. Associate Prof. Per Stål, my assistant supervisor, for your inputs and discussions in science. Your energy in life is inspring. There are for sure lots of people I would like to acknowledge in Anatomy especially: Mona Lindtröm, for being kind and patient whenever I needed your knowledge in lab work. Anna-Karin Olofsson, for introducing me into lab work and ready to answer all my questions. Sture Forsgren, for all your tremendous kindness and hospitality towards me. You indeed taught me a lot and I am grateful for that. Gustav Andersson, for all the afterworks and all the fun we had. Thanks again for the figures. Lars-Eric Thornell, for your comments and sharing your knowedgle. Anton Tjust, for nice atmosphere in lab and all scientific discussions and comments. Anita Dreyer-Perkiömäki, for always being helpful and ready to answer all my questions with patience. Patrik Danielson, for the nice spirit whenever we were having afterworks. Göran Dahlgren, for your help with IT stuff and for reminding me of making back-ups. Anna-Lena Tallander, Selamawit Kefela and Victoria Åström, for helping me with travelling and conferences. All the other former and present colleagues and friends in the IMB Department I met and got to know through these years: Ludvig Backman, Johan Bagge, Peter Boman, Gabor Borbely, Eva Carlsson, Lena Carlsson, Jialin Chen, Rosanna Ching, Catarina Conde, Tushar Devanand Yelhekar, Gloria Fong, Alireza

Acknowledgements

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Janbaz, Amar Karalija, Peyman Kelk, Nausheen Khan, Paul Kingham, Shrikant Kolan, Björn Lindell, Filip Löfgren, Lev Novikov, Ludmila Nvikova, Kevin Peikert, Lina Renström, Farhan Shah, Marta Sloniecka, Wei Zhang, Juha Prittinen and many others … And other friends whom they made my life in Umeå more fun including Parham and Farah for all climbing stuff and gatherings we did together. And of course all other friends I spent time with during these years. External people I would like to appreciate their help: Prof. Homa Tajsharghi, for giving me the opportunity to do a project in your lab in Gothenburg University before starting my PhD. Prof. Linda McLoon, It was very inspiring working on a paper with you. Your energy and enthusiasm amazed me. Associate Prof. Ji-Guo Yu, for your valuable comments on my papers and my thesis. I really appreciate your help. Dr. Susanne Lindquist, for your help and guidance when I was working in your department as a part of my PhD project. Prof. Thomas Brännström and Prof. Peter M. Andersen, for providing material for my studies and your valuable comments throughout my thesis. Ull-Stina Spetz, for taking care of the animals I used in my studies. Dr. Irene Martinez Carrasco, for your help and guidance with confocal microscopy. My family and friends: Christoph my best friend and flatmate, It was an amazing and memorable time for me. We went through all these years and all I will remember will be joy and fun. Thanks for everything mate. Song for helping me a lot especially when I was new in Umeå. I had such nice time with you and your family. Thanks for everything. Sandrine, for organising all outdoor stuff we did together and for all the fun moments we had. Mallappa for all the fun time we had. It was really nice having your company man. Vinay, it was nice having you around. I am happy getting to know your family too. Saeed, for always cheering me up through these years. Mona, for all your kindness and friendship and the theatres you organised. My brother Ali, for always being supportive. I really appreciate all your encouragement and guidance through these years. Afrouz my sister in law, for all your support and kindness in the very begining when I came to Sweden. Navid my nephew, for all the fun we had whenever I was back home. Philippa, my girlfriend, although it wasn’t easy at all to be away from eachother during the last year, but we made it… And finally I would like to thank my mum and dad, my sisters Nahid, Nassrin and Nassim and my brother Mohammad for all your support and kindness.

Fundings

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FUNDINGS

• The county Council of Västerbotten including a Cutting Edege Medical

Research Grant and Central ALF • The Swedish Research Council (K2012-63X-20399-06-3; Dnr 2011-2610) • Karriärbidrag, Umeå universitet • Stiftelsen Kronprinsessan Margaretas Arbetsnämnd för Synskadade • The Swedish Medical Society (SLS) • The Swedish Association for the Neurologically Disabled (NHR) • The Swedish Brain Research Foundation • The Ulla-Carin Lindquist ALS Foundation • Ögonfonden • The Björklund Fond • Berit Hållsten’s Brain Research Foundation

References

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