Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 967
_____________________________ _____________________________
On Pathophysiological Mechanisms in Amyothrophic Lateral Sclerosis
BY
EVA GRUNDSTRÖM
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000
Dissertation for the Degree of Doctor of Medical Science in Neurology presented at Uppsala University in 2000
Abstract
Grundström, E. 2000. On Pathophysiological Mechanisms in Amyotrophic Lateral Sclerosis. Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 967. 52 pp. Uppsala ISBN 91-554-4838-0.
Amyotrophic lateral sclerosis is a fatal, progressive neurodegenerative disease with unknown ethiology. The aim of this study was to increase understanding of the pathophysiological mechanisms of dying motor neurons and wasting muscle tissue in this particular disorder.
Quantitative receptor autoradiographic methodology was applied on cervical spinal cord sections from patients with ALS to evaluate the specific binding of the acetylcholine transporter
3H-vesamicol in motor neurons. Despite a significant reduction of the number of ventral motor neurons in ALS, the 3H-vesamicol binding was not reduced in ALS compared to control cases, which suggests an increased metabolic activity in remaining motor neurons.
Motor neurons dying in ALS might go through apoptosis (programmed cell death), so immunohistochemical and TUNEL techniques were applied on thoracic spinal cord from ALS patients to evaluate the possibility of an apoptotic process. The increased Bax expression indicates an apoptotic process and further, motor neurons were TUNEL-positive, indicating DNA degradation caused by programmed cell death.
Muscle biopsies were obtained from ALS patients, and mRNA levels for the neurotrophic factors GDNF and BDNF were measured and compared to control subjects. GDNF levels were increased in muscle tissue in ALS whereas BDNF levels were unaltered.
Levels of GDNF and BDNF were also measured in cerebrospinal fluid from ALS patients and controls using ELISA methodology. Levels of BDNF were unaltered in ALS compared to controls. GDNF however was not detectable in controls whereas 12 out of 15 ALS patients had measurable levels of GDNF. A marked upregulation of endogenous GDNF and GDNF mRNA in ALS CSF and muscle respectively is of special interest in relation to clinical trials where GDNF is administered to this group of patients.
Key words: Amyotrophic lateral sclerosis, ALS, spinal cord, motor neuron, cerebrospinal fluid, CSF, muscle, glial cell line-derived neurotrophic factor, GDNF, brain-derived neurotrophic factor, BDNF, ACh, vesamicol, apoptosis, Bax, Bcl-2, neurodegeneration.
Eva Grundström, Department of Neuroscience, Neurology, University Hospital, SE-751 85 Uppsala, Sweden
Eva Grundström 2000
ISSN 0282-7476 ISBN 91-554-4838-0
Printed in Sweden by Lindbergs Grafiska, Uppsala 2000
To my mother and to my father
I love the change of seasons the smell of an autumn day in October when one can already feel winter is coming ro
This thesis is based on the following articles, which are referred to in the text by their roman numerals:
I. Grundström E, Gillberg P-G, and Aquilonius S-M.
High 3H-vesamicol binding in ALS motor neurons - autoradiographic visualisation of hyperactivity?
Acta Neurol Scand (2000) In press
II. Ekegren T, Grundström, E, Lindholm D and Aquilonius S-M.
Upregulation of Bax protein and increased DNA degradation in ALS spinal cord motor neurons.
Acta Neurol Scand (1999) 100:317-321
III. Grundström E, Askmark, H, Lindeberg J, Nygren I, Ebendal T and Aquilonius S-M.
Increased expression of glial cell line-derived neurotrophic factor mRNA in muscle biopsies from patients with amyotrophic lateral sclerosis.
J Neurol Sci (1999) 162:169-173
IV. Grundström E, Lindholm D, Johansson A, Blennow K and Askmark H.
GDNF but not BDNF is increased in cerebrospinal fluid in amyotrophic lateral sclerosis.
Neuroreport (2000) 11:1781-1783
Reprints were made with permission from the publishers
ABBREVIATIONS
Ab antibody
ACh acetylcholine
ALS amyotrophic lateral sclerosis Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma /leukemia-2 BDNF brain-derived neurotrophic factor
CSF cerebrospinal fluid
ELISA enzyme linked immuno sorbent assay FALS familial amyotrophic lateral sclerosis GDNF glial cell line-derived neurotrophic factor
ICH-1 ICE and CED-3 homolog-1
LMN lower motor neuron
mRNA messenger ribonucleic acid
RT-PCR reverse transcriptase polymerase chain reaction SALS sporadic amyotrophic lateral sclerosis
TUNEL TdT-mediated dUTP nick end labelling
UMN upper motor neuron
QNB quinuclidinyl benzilate
CONTENT
INTRODUCTION... 9
General Background... 9
Clinical Manifestations ... 9
Epidemiology... 11
Neuropathology... 11
Pathogenesis... 13
Excitotoxicity... 13
Exogenous Toxins... 14
Genetics and FALS... 15
Immune Factors and Autoimmunity... 16
Viral Agents... 16
Apoptosis... 16
Neurotrophic Factors... 21
Neurotrophins... 21
GDNF and Related Proteins... 22
Selective Motor Neuron Vulnerability... 23
Clinical Trials... 24
AIMS OF THE STUDY... 25
MATERIAL AND METHODS... 26
Spinal Cord Tissue (Papers I and II) ... 26
Muscle Biopsies (Paper III) ... 26
Cerebrospinal Fluid Sampling (Paper IV) ... 26
Autoradiography (Paper I) ... 27
Immunohistochemistry (Paper II) ... 27
RT-PCR (Paper III) ... 28
ELISA Methodology (Paper IV) ... 29
RESULTS AND DISCUSSION... 30
CONCLUSION... 33
FUTURE PROSPECTS... 34
ACKNOWLEDGEMENTS... 35
REFERENCES... 39
INTRODUCTION
General Background
The term amyotrophic lateral sclerosis (ALS) was first proposed by the French neurologist Jean-Martin Charcot in 1869 (Charcot and Joffroy, 1869). He and Joffroy described the pathological changes in the spinal cord of patients who had died from a disorder presenting asymmetric progressive wasting of the skeletal muscles, fibrillary contractions and fatigue. These findings of characteristic involvement of the corticospinal tract and loss of motor neurons had been preceeded by several closely related findings made by other physicians. Aran coined the term progressive muscular atrophy (PMA) after describing patients with muscle wasting in one or several limbs (Aran, 1850) and Duchenne described patients with rapidly progressive atrophy of the bulbar innervated muscles, and proposed the term progressive bulbar palsy (PBP) (Duchenne, 1860).
Primary lateral sclerosis (PLS), first described by Erb, is a rare neuromuscular disorder, primarily affecting the upper motor neurons (Erb, 1875) (Figure 1).
Brain introduced the term motor neuron disease (MND) as a generic name for ALS, PMA and PBP as they all present a spectrum of upper motor neuron (UMN) and lower motor neuron (LMN) involvement resulting in focal and general muscular wasting (Brain, 1962).
UMN<--->LMN
PLS PBP ALS PMA
Figure 1. The clinical spectrum of motor neuron diseases.
Clinical Manifestations
ALS is a relentlessly progressive, invariably fatal neurological disease, that is characterized clinically by progressive muscle weakness, muscle atrophy and paralysis. The neuropathology of ALS is primary degeneration of the upper (motor cortical) and lower (brainstem and spinal) motor neurons. The amyotrophy refers to the neurogenic atrophy of affected muscle groups and
the lateral sclerosis refers to the hardening of the lateral white matter in the spinal cord. Death typically results from respiratory failure and the mean span from disease onset to death is typically 3-5 years (Kuncl et al., 1992).
The varying extent and localization of involvement of the motor system results in different clinical features, but ultimately, as the disorder progresses, the clinical expression of the disease is rather uniform, with extreme muscular atrophy and spasticity.
The clinical features depend on the combination of UMN and LMN involvement, and these vary not only according to the pattern of onset but also in relation to the stage of the disease. LMN involvement causes weakness and fatigue, muscle wasting, fibrillation and fasciculation, hyporeflexia and muscle cramps. UMN involvement causes weakness, incoordination, spasticity and hyperflexia. These features are present in varying combinations in the different body segments or regions. Obvious symptomatic weakness and the objective demonstration of loss of muscle strength require considerable loss of functioning motor neurons. Usually as many as 50-80% of motor neurons have been lost when weakness is detected on examination (Wohlfart, 1957). This is explained by a compensatory reinnervation process in which surviving motor neurons sprout in a collateral fashion to maintain innervation of adjacent muscle fibers in the partially denervated muscle. As a result of collateral sprouting, remaining motor neurons tend to grow in size. Although ALS presents as a disease of the motor system, terminally ill patients may present a multisystem degeneration of Clark’s dorsal nuclei, the spinocerebellar tracts (Swash et al., 1998), the dorsal column of sensory pathways (Ince et al., 1996), substantia nigra (Murakami et al., 1995; Kato et al., 1993;
Mizutani et al., 1992) as well as frontal function changes (Abrahams et al., 1997; Rakowicz and Hodges, 1998; Chari et al., 1996).
ALS debuts either as a limb onset (65-80% of the cases) or bulbar onset (20-25% of the cases). The typical limb onset presents an asymmetric distal weakness and atrophy of a hand or foot. The patient might trip over or drag one foot or have problems with fine movements of a hand. Bulbar features develop during the course of the disease, but may be a presenting feature, especially in middle-aged women with ALS. Bulbar involvement typically includes difficulty swallowing and speaking, and fasciculation of the tongue, and is often associated with weakened respiratory muscles. Thus, initial bulbar involvement carries a poor prognosis and life expectancy in
patients with bulbar ALS is, according to a study by Gubbay and colleagues (Gubbay et al., 1985), less than in those who present with limb onset.
Epidemiology
The incidence of ALS is about 1-2 cases per 100,000 inhabitants per year.
It has a prevalence of about 4-9 per 100,000, a figure directly dependent on the survival of those affected. The incidence is fairly similar throughout the world with a few exceptions with higher frequency, such as Guam (Reed et al., 1975), the Kii Peninsula of Japan (Kimura, 1965) and the southern lowlands of western New Guinea (Gajdusek and Salazar, 1982). There is controversy as to whether the disease is increasing in incidence or whether it is being more accurately diagnosed than previously (Gunnarsson et al., 1990; Swash and Schwartz, 1997; Li et al., 1990). Clearly, diagnostic accuracy is very important in assessing changes in incidence of ALS. The disease typically presents in the later half of life, between 45–60 years of age (Friedman and Freedman, 1950; Mackay, 1963), but younger cases are also found (Ben Hamida et al., 1990). There is a slight overrepresentation of men with ALS compared to women (1.6:1). A subgroup of approximately 5-10% of ALS patients has been found to have hereditary forms and are thus designated familial ALS (FALS). The mode of inheritance and penetrance is not fully understood.
Neuropathology
The major pathological features in ALS, with both UMN and LMN degeneration, are a reduction in both the number and size of lower motor neurons (anterior horn cells) in the spinal ventral horns and bulbar motor nuclei, and myelin pallor in the corticospinal projection pathway, a secondary consequence of axonal loss in this region. In severely affected cases, loss of UMN (Betz cell) can be detected in the motor cortex area (Brownell et al.,1970). Two motor neuron pathways are selectively spared in ALS; the sacral Onuf’s nuclei (Mannen et al., 1977; Iwata et al., 1978) and the motor neurons of the third, fourth and sixth cranial nerves, which innervate the external ocular muscles (Hughes, 1982). Hence, ALS is typically associated with retention of urinary and fecal continence and normal eye movements, even at very late stages of the disease where most other muscle groups are severely affected. Other obvious pathological signs
are muscle denervation atrophy (Martin and Swash, 1995) due to a series of denervation–reinnervation-denervation events. The reinnervation arises from collateral sprouting of intramuscular axons (Wohlfart, 1957).
Different types of intraneuronal inclusion bodies in anterior horn cells in ALS have been found:
Ubiquinated Inclusions
Ubiquitin is a 76 amino acid polypeptide and its best known role at present is in the non-lysosomal degradation of short-lived and damaged proteins (Hochstrasser, 1995). Ubiquinated inclusions (UBI) are found in 80-100% of sporadic ALS (SALS) cases (Leigh et al., 1991). Although many of the inclusion bodies of neurodegenerative diesases, including ALS, are heavily ubiquitinated there is no evidence for any primary abnormality of the ubiquitin-proteasome pathway in ALS. Rather, it appears that ubiquitination is a consequence of the accumulation of abnormal proteins despite the availability of pathways for protein degradation.
Bunina Bodies
Bunina bodies are small (1-4 µm) round eosinophilic inclusions, located in the cytoplasm of the perikarya. They have been described in both SALS and FALS (Bunina, 1962) and are reported to be present in 30-50% of cases. Bunina bodies are most commonly found in patients with ALS but they have also been described in other closely related disorders (Hayashi et al., 1997; Semmekrot et al., 1998).
Hyaline Conglomerate Inclusions
Hyaline conglomerate inclusions (HCI) consist of large aggregates of 10-15 nm diameter neurofilaments. They are not ALS specific but have been described both in controls and patients with other neurological diseases (Leigh et al., 1989; Sobue et al., 1990).
Neurofilaments
Neurofilaments are the major class of cytoskeletal intermediate (10 nm) filaments that characterize neurons. Abnormal accumulation of neurofilaments is a common finding in several types of neuro- degenerative diseases, including SALS (Carpenter, 1968; Hirano, 1991) and FALS (Hirano et al., 1984). Slow axonal transport is associated with the presence of medium and heavy chain
neurofilaments, which characterize large neurons, such as motor neurons.
Globules and Spheroids
Axonal swellings in the anterior horn have been described in ALS spinal cord. These spheriods and globules are composed of abnormally orientated aggregations of 10 nm neurofilaments (Robinson and Anderton, 1988; Carpenter, 1968). Both the larger spheriods (20-100 µm) and the smaller globules are present in control cases and hence have no disease specificity. Spheroids are commonly found in proximal axons close to motor neuron somata, whereas globules tend to be more peripheral in the ventral horn. The number of spheroids is increased in ALS (Leigh et al., 1989; Sobue et al., 1990;
Manetto et al., 1988), and it seems they accumulate as a result of the slowed axonal transport.
Pathogenesis
Many etiological hypotheses have been brought forward to account for the relatively selective degeneration of motor neurons in ALS. None of current hypotheses can fully explain the selective loss of anterior horn cells, and it is likely that ALS is caused by a variety of primary insults, all of which culminate in the final clinical phenotype of UMN and LNM degeneration characteristic of ALS. This multiplicity of etiologies for the disease progress is most convincingly illustrated by the familial variants of ALS, in which some families possess linkage to mutations in the copper-zink superoxide dismutase gene (SOD1), while others do not (Strong et al., 1991; Rosen et al., 1993) and the striking similarities in clinical phenotype between the sporadic and familial form of ALS.
Excitotoxicity
The term excitotoxicity was coined to describe the neurodegeneration resulting from exposure to excitatory amino acids. Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS) and its neurotoxic properties were first discovered in the late 1950’s (Lucas and Newhouse, 1957). The first evidence that glutamate could be related to ALS came when Plaitakis and Caroscio revealed increased levels of glutamate in plasma from patients with the disease (Plaitakis and Caroscio,
1987). Another excitatory amino acid, aspartate, was also found to be increased in the same study. Increased glutamate levels have also been found in cerebrospinal fluid (CSF) from ALS patients (Rothstein et al., 1990; Shaw et al., 1995). Interestingly, other amino acids were not elevated in ALS. These findings led to the hypothesis of a possible defect in glutamate transport, since aspartate and glutamate exclusively are removed from the extracellular space by the same transport system. Further studies revealed a glutamate transport deficiency in the motor cortex and spinal cord in ALS patients compared to controls (Rothstein et al., 1992).
Rothstein then tested this hypothesis in rat organotypic spinal cord cultures where he added drugs that non-selectively blocked all glutamate transport subtypes. This chronic blockage of glutamate transport in vitro led to an increased concentration of cellular glutamate and a slow selective loss of motor neurons, which was shown to be prevented by co-administration of specific glutamate receptor antagonists (Rothstein et al., 1993). Rothstein and co-workers also found decreased immunoreactivity for glutamate transporter–1 (GLT-1), also known as excitatory amino acid transporter 2 (EAAT2), in ALS motor cortex and spinal cord (Rothstein et al., 1995).
Continuous studies in knock-out rats revealed a selective vulnerability for the lack of the astroglial glutamate transporters EAAT1 and EAAT2, resulting in neurodegeneration and progressive paralysis beginning in the hind limbs in this knock-out model (Rothstein et al., 1996). These results indicate that the main portion of glutamate clearance occurs through the astroglial transporters. Immunoprecipitation studies showed that EAAT2 is the dominant glutamate transporter in the brain (Haugeto et al., 1996).
These results indicate that the increase in CSF glutamate levels and the impaired loss of tissue glutamate transport in ALS patients are due to selective loss of the EAAT2 protein. Lin and co-workers found abnormal mRNA coding for the EAAT2 protein in 65% of patients with SALS (Lin et al.,1998). Their study indicated that these mRNA species undergo rapid degradation and also exert a negative effect on the wild type EAAT2 mRNA, resulting in the loss of ”normal” protein activity. The positive effect on the outcome of ALS when administrating the glutamatergic transmission blocker, Riluzole, also supports the excitotoxicity hypothesis.
Exogenous Neurotoxins
ALS increases in incidence with age. It can be argued that the elderly are at greatest risk because of cumulative exposure to an environmental agent.
Many possible exogenous neurotoxins have been proposed, including exposure to lead and mercury (Felmus et al., 1976), milk ingestion and athletic activity (Felmus et al., 1976), solvent exposure (Hawkes et al., 1989), mechanical trauma (Bracco et al., 1979; Kondo and Tsubaki, 1981;
Sienko et al., 1990), welding or soldering exposure (Strickland et al., 1996), exposure to plastics (Deapen and Henderson, 1986), electrical insult (Gawel et al., 1983; Deapen and Henderson, 1986; Schulte et al., 1996), aluminium exposure, calcium and magnesium deficiency (Garruto et al., 1983; Kimura et al., 1963) and cycas circinalis intoxication (Spencer et al., 1987). The resolution of ALS amongst the inhabitants of Guam and the linkage to environmental agents is striking evidence that ALS can be associated with an environmental trigger. It still remains to be determined what the causative agent(s) are that gave rise to ALS in this focus. A multi- insult process, in which one or more environmental agents play a major role, is the most probable explanation.
Genetics and FALS
In a subset of ALS cases, the disease is apparently inherited as an autosomal, dominant trait with high penetrance (Emery et al., 1982; Mulder et al., 1986). The realization that ALS could be inherited dates back to the earliest descriptions of the disease (Aran, 1850). The inheritance hypothesis was however not generally accepted until 1955, when Kurland and Mulder reported six new families with FALS (Kurland and Mulder, 1955).
Epidemiological studies have revealed a FALS frequency of 5-10% in most populations but FALS is probably under diagnosed due to difficulties to establish family histories, and families with low disease penetrance. In 1991 Siddique and colleagues reported finding linkage to chromosome 21 for some FALS families (Siddique et al., 1991). Mutations in the SOD1 gene on chromosome 21 were later found to be present in about 15–23% of families with FALS (Rosen et al., 1993; Siddique et al., 1996). The SOD1 enzyme catalyses the conversion of superoxide free radical anions to hydrogen peroxide and dioxygen, but the neurotoxic mechanism by which the mutant SOD1 enzyme causes motor neuron death is still not fully understood. The accumulated studies suggest that a loss of SOD1 activity is not the cause of neurodegeneration in ALS, but rather points towards an acquired cytotoxic function for the mutated SOD1. Well over 60 different mutations have been found in the SOD1 gene in FALS patients. These are widely scattered throughout the gene (Radunovic and Leigh, 1996). Despite
the variety of mutations, there are no obvious clinical differences between the different classes of mutations. Moreover, FALS patients with SOD1 mutations are clinically indistinguishable from FALS or SALS patients without a known SOD1 mutation (Cudkowicz et al., 1996; Orrell et al., 1997).
Immune Factors and Autoimmunity
Autoimmunity or immunological factors contributing to the disease process in ALS have been debated for more than 30 years. Evidence of immunological involvement in ALS is a higher incidence of immune disorders in patients with ALS, such as the presence of paraproteinemias (5-10%) (Shy et al., 1986; Younger et al., 1990) and lymphomas (Younger et al., 1991) in ALS cases, the finding of IgG within ALS motor neurons (Engelhardt and Appel, 1990; Fishman and Drachman, 1995) and the presence of inflammatory cells (Engelhardt and Appel, 1990; Engelhardt et al., 1993). Despite these findings one can argue the importance of the autoimmune hypothesis in ALS since immunosuppressive treatments have failed to alter the outcome of the disease.
Viral Agents
Conflicting reports on the relationship between chronic enterovirus (EV) infections and ALS have been published (Leparc-Goffart et al., 1996;
Swanson et al., 1995; Muir et al., 1996). New findings suggest that this hypothesis should be considered seriously (Berger et al., 2000). Thus, this group has detected enteroviral sequences in the neuronal cell bodies within the gray matter in 15 out of 17 ALS spinal cord samples, in comparison to 1 out of 29 control subjects. The authors strongly suggest an association between persistent enterovirus RNA and ALS.
Apoptosis
Cell death can occur by different pathways. Depending on the type of cell and on the stimulus, cells die typically in either of two ways, generally described as apoptosis (Figure 2) or necrosis (Martin et al., 1998; Kerr et al., 1972). Apoptosis is a mode of structurally organized cell death in which the cell actively participates in its own destruction by activating a pre-programmed intracellular suicide machinery. Hence, apoptosis is also
known as programmed cell death (PCD). Morphological changes in apoptosis include chromatin condensation, cytoplasmic vacuolization, cell shrinkge, and plasma membrane blebbing followed by formation of membrane-enclosed apoptotic bodies (Kerr et al., 1972; Bredesen, 1995).
Figure 2. Schematic figure of the phases of the apoptotic process.
Cell cycle arrest
Capacitation for apoptosis or proliferation
Irreversible committment
to death
”pre-apoptosis”
Apoptosis:
Nucleolysis Chromatinolysis
Proteolysis Cytolysis
Induction phase
Effector phase
Degradation phase
Bax
ICH-1L
Bcl-2 Growth
factor withdrawal
DNA damage
At the biochemical level, apoptosis is often, associated with nuclear DNA fragmentation (Wyllie, 1980). In contrast, necrosis is a more rapid and random degeneration, resulting from abrupt pathophysiological perturbations such as osmotic, toxic, thermal or traumatic incidences, and involving disruption of plasma membrane, rapid influx of Na+, Ca2+ and H2O and a failure of cell volume homeostasis.
Apoptosis is mediated through several different pathways, depending on the initial stimulus. Three families of apoptosis regulating genes have been described in mammals: the Bcl-2 family (Merry and Korsmeyer, 1997), the IAP family (LaCasse et al., 1998), and the caspases (Schwartz and Milligan, 1996; Wolf and Green, 1999).
The Bcl-2 family members can be divided into two groups according to their function: the antiapoptotic members (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1) and the proapoptotic members (Bax, Bak, Bok, Bcl-XS, Bad, Bid, Bik/Nbk, Bim, Krk, Mtd) (Antonsson and Martinou, 2000). Membership of the family of Bcl-2-related proteins is defined by Bcl-2 homology (BH) domains (BH1-BH4), which function in the interactions between members.
BH1 and BH2 seem to be necessary for antiapoptotic activity (Borner et al., 1994), whereas BH3 seems to trigger apoptosis (Hunter and Parslow, 1996). Bcl-2 family members exist as monomers that form homodimers and heterodimers and higher order multimers (Merry and Korsmeyer, 1997), but they can also function independently to regulate cell survival (Cheng et al., 1996). Bax (Bcl-2-associated X protein) can act both as a homodimer and as a heterodimer. In the homodimeric state, Bax functions as a cell death promoter, whereas in the heterodimeric state, attached to Bcl-2 (B-cell lymphoma /leukemia-2), it represses Bcl-2’s antiapoptotic effect. This lead to a model in which the ratio of Bcl-2 to Bax determines if cells will survive or die from an apoptotic stimulus (Mu et al., 1996;
Korsmeyer et al., 1993; Vekrellis et al., 1997).
ICH-1 (ICE and CED-3 homolog) is a gene related to the C. elegans ced-3 and its mammalian homologue interleukin-1β-converting enzyme (ICE).
ICH-1 belongs to the interleukin-1β-converting enzyme (ICE)-like cysteine proteases, which are also known as caspases. These proteases are translated as inactive proenzymes that, once activated, cleave their substrates after aspartate residues. Two distinct mRNA species of ICH-1 have been found, ICH-1L and ICH-1S. Overexpression of ICH-1 L has been found to induce
apoptosis whereas ICH-1S seems to have a preventive effect on apoptosis (Wang et al., 1994).
Apoptosis is also regulated by the IAP (inhibitor of apoptosis protein) family. This family consists of X-chromosome-linked IAP, IAP1, IAP2 and NAIP (neuronal apoptosis inhibitory protein). IAPs are thought to supress apoptosis by preventing caspase activity (LaCasse et al., 1998; Deveraux et al., 1998).
Several reports have described evidence of an apoptotic process in tissue from patients with ALS (for review, see Martin et al., 2000). Yoshiyama and colleagues identified the expression of an apoptosis-related antigen, Le(Y), in seven out of ten ALS cervical spinal cord samples (Yoshiyama et al., 1994). In these seven samples, apoptosis was also confirmed by TUNEL technique detecting DNA fragmentation. DNA fragmentation in ALS has also been detected by other groups (Migheli et al., 1994; Troost et al., 1995; Ekegren et al., 1999 in the present thesis). This method does not conclusively rule out the possibility of necrotic death of a neuron, but recent morphological findings by Martin support the hypothesis of an apoptotic cell death of motor neurons in ALS (Martin, 1999). Martin’s report indicated that neuronal death in ALS has all the characteristics of apoptosis as revealed by signs of typical chromatolysis in spinal cords of ALS patients and increased Bax and decreased Bcl-2 expression in the mitochondria-enriched membrane compartment of vulnerable regions.
Troost and co-workers detected cell shrinkage and small Nissl-positive bodies in the ventral spinal cord of ALS patients (Troost et al., 1995). In addition, immunostaining showed an identical distribution and unaltered expression of Bcl-2 protein between ALS and non-neurological controls.
Mu and colleagues used in situ hybridization to analyze bcl-2 and bax mRNA levels in control and ALS lumbar spinal cord sections (Mu et al., 1996). Compared with controls, bcl-2 mRNA levels were lower in ALS whereas bax mRNA levels were higher. Interestingly, bax mRNA expression was selectively increased in ALS motor neurons.
The IAP family of antiapoptotic molecules has not been closely studied in relation to ALS except for a recent paper by Pari and co-workers. They investigated the expression of the protein NAIP in spinal cord and motor cortex in ALS cases and controls but found no difference in expression between the two groups studied (Pari et al., 2000).
Receptor on motor neurons Neurotrophins Brain-derived neurotrophic factor (BDNF)p75NTR , trk-B Neurotrophin-3 (NT-3)p75 NTR , trk-C Neurotrophin-4/5 (NT-4/5)p75 NTR , trk-B Ciliary Neurotrophic Factor (CNTF) – Leukemia Inhibitory Factor (LIF) Family CNTFCNTF receptor-α, LIF receptor-β gp130 LIFLIF receptor-β gp130 Cardiotrophin-1 (CT-1)LIF receptor-β gp130 Hepatocyte Growth Factor (HGF)c-met Insulin-like Growth Factors (IGF’s) IGF-IIGF receptor-1 IGF-IIIGF receptor-1, mannose-6-phosphate receptor Glial cell line-derived neurotrophic factor (GDNF) and Related Factors GDNFRet, GFR α-1 Neurturin (NTN)Ret, GFR α-2 Artemin (ART)Ret, GFR α-3 Persephin (PSP)Ret, GFR α-4 Table 1. Neurotrophic factors for motor neurons and their receptors
Neurotrophic Factors
Studies on trophic factors and an appreciation of their importance during neuronal development started decades ago when Levi-Montalcini and Hamburger showed that nerve growth factor (NGF) was responsible for neuronal growth (Levi-Montalcini and Hamburger, 1953). During development, motor neurons are generated in excess and ultimately, as the neurons make contact with their target tissue, the skeletal muscle, approximately 50% of them die through a process known as ”physiological motor neuron cell death”. Motor neuron neurogenesis and post-natal survival are dependant upon target-derived neurotrophic molecules, such as neurotrophins (BDNF, NT-3 and NT-4/5), members of the TGF-b- superfamily (GDNF, NTN, PSP and ART), neurokines (CNTF, LIF and cardiotrophin), insulin-like growth factors (IGF’s) and hepatocyte growth factor (HGF) (see Table 1 for motor neuron specific neurotrophic factors).
Neurotrophins
The neurotrophins constitute a family of four proteins in mammals, NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 and 4/5 (NT- 3 and NT 4/5). These neurotrophins all bind to a common low-affinity receptor, p75NTR, but at different binding sites (Chao and Hempstead, 1995). They also bind to specific high-affinity receptors: trk-A (specific for NGF), trk-B (specific for BDNF and NT-4/5) and trk-C (specific for NT-3).
BDNF has been shown to promote survival of developing motor neurons (Henderson et al., 1993; Wong et al., 1993) and has also been reported to rescue developing motor neurons from axotomy-induced (Yan et al., 1992;
Sendtner et al., 1992; Koliatsos et al., 1993; Clatterbuck et al., 1994) or naturally occurring cell death (Oppenheim et al., 1992). BDNF mRNA is expressed in adult rat muscle and increases following axotomy (Koliatsos et al., 1993; Funakoshi et al., 1993). BDNF mRNA is also expressed in the adult sciatic nerve and the level increases dramatically in the distal segment after axotomy of the sciatic nerve (Funakoshi et al., 1993; Meyer et al., 1992). Furthermore, axotomized adult motor neurons respond to BDNF (Yan et al., 1994). Recently, Kawamoto and colleagues performed a study localizing BDNF in ALS spinal cord sections (Kawamoto et al., 1998).
They observed immunoreactivity for the BDNF protein in remaining motor neurons in ALS at a similar magnitude as seen in the control cases. BDNF was also found to be localized in non-motor neurons within the spinal cord.
Both Trk-B protein and mRNA expression have been shown to be elevated in ALS spinal cord (Mutoh et al., 2000). Despite an up regulation of Trk-B protein in ALS spinal cord its intracellular signal transduction pathway might be less active compared to controls since the tyrosine phosphorylation levels are reduced. Taken together these findings suggest that motor neurons in ALS patients may receive a sufficient BDNF supply from surrounding neurons, whether non-motor neurons or non-degenerated motor neurons. This conclusion is also supported by clinical trials in which BDNF administrered to ALS patients did not alter the outcome of the disease (The BDNF Study Group, 1999).
Glial-Derived Neurotrophic Factor (GDNF) and Related Factors
This group of neurotrophic factors was initially discovered in 1993 when the gene for GDNF was cloned (Lin et al., 1993). At present, the family of GDNF related proteins constitutes of neurturin (NTN) (Kotzbauer et al., 1996), persephin (PSP) (Milbrandt et al., 1998) and artemin (ART) (Baloh et al., 1998). These members of the GDNF family have seven conserved cysteine residues with similar spacing, making them distant members of the transforming growth factor-beta (TGF-beta) superfamily. GDNF, NTN, PSP and ART mediate their signals via a common receptor tyrosine kinase, Ret (Trupp et al., 1996; Mason, 2000), but their ligand specificity is determined by a novel class of glycosylphosphatidylinositol (GPI)- anchored proteins called the GDNF family receptor alpha (GFR α) (Treanor et al., 1996; Jing et al., 1996). GDNF binds preferentially to GFR α-1, NTN to GFR α-2, ART to GFR α-3 and PSP to GFR α-4 as a co- receptor to activate Ret. (Summarized in Baloh et al., 2000).
GDNF has been shown to be a very potent factor in supporting motor neuron survival in experimental models (Lin et al., 1993; Henderson et al., 1994). In vivo, GDNF prevents programmed cell death of motor neurons during ontogenesis (Oppenheim et al., 1995) and axotomy-induced degeneration of motor neurons (Yan et al., 1995). Yamamoto and colleagues measured the levels of GDNF in post-mortem ALS muscle and spinal cord and found decreased expression in muscle and increased levels in spinal cord in comparison to controls (Yamamoto et al., 1996). More recent studies in ALS muscle however, revealed an increased expression of GDNF mRNA compared to controls (Lie and Weis, 1998; Yamamoto et al., 1999; Grundström et al., 1999 in the present thesis). Immunostaining for the Ret receptor showed a persistant expression of the Ret protein in
remaining motor neurons in ALS spinal cord as compared to control cases (Duberley et al., 1998). Mitsuma and co-workers also reported on a preserved GDNF receptor expression in an investigation of both mRNA and protein levels of GFR α-1 and Ret in ALS spinal cord. Ret mRNA and protein were exclusively expressed in motor neurons whereas GFR α-1 was expressed in both motor neurons and glial cells (Mitsuma et al., 1999). A study carried out in muscle tissue from patients with ALS revealed an unaltered expression of GFR α-1 mRNA in comparison to controls (Yamamoto et al., 1999). The same group failed to detect Ret mRNA in muscle tissue from ALS patients and control cases.
Motor Neuron Vulnerability
Motor neurons are one of the largest types of neurons and they also have very long axons. For maintenance purposes they rely upon axonal transportation, which requires a high level of mitochondrial activity, a high metabolic rate and a high neurofilament content. This transportation has a high energy demand, and hence motor neurons tend to be sensitive to oxidative stress, resulting in neurofilament accumulation in the axons. This theory is supported by the fact that the class of motor neurons dying at the highest frequency in ALS are the very large-calibered ones with the highest neurofilament content.
Motor neurons affected in ALS, in comparison to other neurons do not contain the calcium buffering proteins, calbindin D-28K and parvalbumin (Alexianu et al., 1994; Shaw and Eggett, 2000). Motor neurons which are selectively spared in ALS (the oculomotor nuclei and Onuf’s nucleus) are potentially protected by their calcium-buffering properties. Motor neurons contain a high proportion of the AMPA-kainate receptor (Rothstein et al., 1992) but lack the GluR2 AMPA-kainate receptor subunit (Williams et al., 1997). The presence of this specific receptor subunit determines the calcium permeability of the receptor and its absence allows for calcium influx in the postsynaptic cell. These findings might mean that motor neurons are particularly vulnerable to calcium toxicity following glutamate receptor activation. This might result in an increased sensitivity for excessive glutamate levels.
Motor neurons normally have a very high content of SOD1 protein (Tsuda et al., 1994) compared to other cells in the nervous system. One
explanation might be that this reflects the importance for motor neurons defending themselves against oxidative stress. This requirement may however make motor neurons more vulnerable to injury in the presence of mutant SOD1 proteins.
Clinical studies
A wide variety of pharmaceutical agents have been tested to try to modify the course and outcome of ALS. The results of almost all earlier trials have been considered to be negative (Askmark and Aquilonius, 1994). As a consequence, both the compounds tested and the putative pathogenic mechanisms were discarded.
Riluzole is at present the only available disease modifying drug for ALS.
The drug supposedly blocks glutamatergic neurotransmission in a syngergistic manner in CNS (Martin et al., 1993) by activating a G-protein- dependent process that leads to inhibition of glutamate acid release and of the mobilization of calcium. The effect is however modest, yet increasing the survival time for ALS patients (Louvel et al., 1997; Lacomblez et al., 1996).
Several clinical trials with administration of neurotrophic factors (CNTF, IGF-I, BDNF, GDNF) have been, or are still being carried out. At present, treatment with CNTF (ALS CNTF Treatment Study Group, 1996; Miller et al., 1996), IGF-1 (Lai et al., 1997) and BDNF (The BDNF Study Group, 1999) have turned out negative, failing to alter the outcome in a positive manner. Clinical trials administrating GDNF are still ongoing.
AIMS OF THE STUDY
General
The aim of this study was to investigate different pathophysiological mechanisms in an attempt to reach a further understanding of the etiology and mechanisms contributing to the disease process of ALS.
Specific
To investigate if 3H-vesamicol could act as a marker of metabolic activity in the remaining motor neurons in ALS (Paper I).
To further investigate a possible apoptotic process in motor neurons in ALS by studying the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax and by studying DNA fragmentation in ALS spinal cord sections (Paper II).
To measure mRNA levels of two important survival factors for motor neurons, GDNF and BDNF, in muscle biopsies from patients with ALS (Paper III).
To measure protein levels of GDNF and BDNF in CSF from patients with ALS in relation to the previous study in muscle tissue (Paper IV).
MATERIAL AND METHODS
All ALS patients included in our studies have been diagnosed according to the El Escorial Criteria (Brooks et al., 1994) and they all possess the sporadic form of the disease (SALS). Further clinical data is found in Papers I – IV.
Spinal Cord Tissue Preservation and Preparation (Papers I and II) Cervical and thoracic spinal cord segments were removed at autopsy and sliced into 5 mm segments after removal of dura mater,. The segments were then immediately frozen on a metal plate maintained in liquid nitrogen and stored at -70°C until experiments were performed. Cervical and thoracic segments were sliced into 10 and 8 µm thick sections respectively on a cryomicrotome (Reichert-Jung 2800 Frigocut, Germany) and mounted on chrome-alum gelatine coated slides and poly-L-lysin coated slides respectively, and thereafter placed in a desiccator overnight at –20°C. Sections were stored at –20°C until incubations and analysis were performed.
Muscle Tissue Collection (Paper III)
Muscle biopsies were performed using the conchotome technique (Henriksson, 1979) and tissue was taken from the deltoideus, the tibialis anterior or the vastus lateralis muscle. Manual muscle testing was made according to the Medical Research Council (MRC) scale (Medical Research Council, 1986) and a moderately weak muscle (grade 3 -4) was chosen in each ALS patient. A typical muscle biopsy procedure resulted in 100 mg tissue. The tissue was immediately frozen under sterile conditions in liquid nitrogen and stored at -150 °C until RNA isolation was performed.
Cerebrospinal Fluid Sampling (Paper IV)
Cerebrospinal fluid was obtained by lumbar puncture. The control CSF samples were collected prior to spinal anesthesia followed by orthopaedic surgery, due to arthrosis of the hip or knee. All CSF samples were stored at -70°C before the study was performed.
Autoradiography (Paper I)
Receptor autoradiography (Palacios et al., 1981) has the ability to both localize and quantify ligand binding and enable the examination of receptor regulation in disease processes. We wanted to apply the receptor autoradiography technique in cervical spinal cord sections from ALS patients and control cases to further investigate the cholinergic motor neurons that specifically die off in ALS. We used the ACh transporter, 3H- vesamicol, as a putative ligand for synaptic vesicle membranes in motor neurons and compared the binding pattern to that of 3H-QNB, a muscarinic receptor ligand.
All sections were preincubated in buffer (for further details see Paper I), 4 x 15 min at 25°C. Thereafter, sections were incubated for 60 min at 23 - 25°C in buffer containing 3H-labelled ligand and parallel sections were co- incubated with a blocker to obtain non-specific binding. Sections were then rinsed in buffer with time and temperature parameters depending on the ligand used. Finally, all sections were dipped in water, dried in a stream of air and stored with a desiccant at -20°C before application of 3H-sensitive film. A typical exposure for 3-6 weeks gave optimal tissue resolution compared to non-specific section densities. Quantitative image analysis was performed for the two ligands using Optilab/Pro 2.5.1 software and gray values were converted to concentrations (fmol/mg wet weight) by means of calibrated standards co-exposed with the sections. Statistical analyses were performed using ANOVA followed by post hoc comparison using Fisher’s protected least square difference test (Fishers PLSD).
Immunohistochemistry (Paper II)
Three series of slides were prepared for immunohistochemical tests, one serie for Bcl-2, Bax and ICH-1L respectively, with three adjacent spinal cord sections in each. One additional series of slides was cut for the TUNEL stainings.
The first section in each series was stained with cresyl violet for morphological identification of nerve cells. The second section was stained with primary antibodies (Ab) specific to the acetylcholine synthesizing enzyme, choline acetyl-transferase (ChAT), for detection of cholinergic motor neurons (Aquilonius et al., 1981; Fonnum, 1973). The third section was double-stained with primary Ab specific to ChAT, together with Ab specific to either Bcl-2, Bax or ICH-1 . For visualization, fluorescent-
labeled secondary Ab were used. Sections were thawed and fixed in 4%
paraformaldehyde. Sections were blocked and a primary Ab was added and slides incubated at 4°C overnight. Sections were incubated with a secondary Ab for 90 minutes at room temperature. Finally, coverslips were applied. Sections from each ALS and control case were also tested with the TUNEL-technique and stained with diaminobenzidine. TUNEL-staining and conversion of TUNEL-positive cells by an anti-fluorescein-antibody were performed according to the product description for TUNEL-POD.
Statistical analyses were performed with Student's t-test for unpaired samples and the Mann-Whitney U-test.
RT-PCR (Paper III)
Total RNA purification was performed as described in the Ultraspec II RNA isolation system kit (Biotecx, US). Typically 100 mg tissue gave 30 - 40 µg total RNA. To determine the total RNA yield we measured OD spectrophotometrically at 260 nm. 3 µg total RNA was used for each cDNA synthesis. 1 µl 50 µM random primer, 3 µg total RNA and distilled water to a final volume of 18.3 µl were incubated at +25 °C for 10 min.
Thereafter, a 2 min incubation at 42 °C was performed with an additional mixture of 5x first strand buffer, 26.7 mM DTT, 1.3 mM dNTP and 20 U rRNAsin. 200 U Superscript was added followed by an incubation for 50 min at 42°C. Finally, a 15 min heat inactivation at 70 °C was performed.
Primers for GDNF and BDNF were designed to detect cDNA, coding for the mature proteins, of a length of 292bp and 339bp respectively.
The PCR reactions were run in 26 cycles of 45 sec at 94 °C followed by 30 sec at 55°C and finally 90 sec at 72°C. The PCR mix contained buffer + Mg2+, 1:10, 300 µM dNTP, 20 pmol of each primer, 2.5 U TAC, 1.5 µM [R110] dUTP, 1 µl cDNA and distilled water to a final volume of 25 µl.
PCR products were kept in the dark at 4°C until they were run on a 6%
polyacrylamide gel on an ABI373 automatic sequencing machine. Each sample gave a fluorescent peak of the correct size that was quantified using the GeneScan 672 software. Statistical analysis was performed using Students t-test for unpaired samples.
ELISA Methodology (Paper IV)
We used a sensitive two-step sandwich ELISA methodology for both neurotrophic factors measured. Plates were coated overnight at 4°C with primary anti-BDNF/anti-GDNF Ab. Next, wells were blocked for 4 hours at room temperature for BDNF and 1 hour for GDNF. The samples and standards (concentration 0.1 – 256 pg/well and 16 - 1000 pg/well for BDNF and GDNF, respectively) were applied in triplicate and duplicate respectively and incubated overnight or 6 hours at room temperature. After washing, a secondary Ab was added and incubation was carried out overnight. Next, a three-hour incubation with avidin-β- galactosidase Anti- Chicken IgY and a two-hour incubation with HRP Conjugate was carried out at room temperature for BDNF and GDNF, respectively. Finally, a 4- methylumbelliferyl-β-galactosidase and MgCl2 buffer was added to the BDNF plates and the amount of fluorescence was measured using a fluorometer. To the GDNF plates, TMB and Peroxidase Substrate were applied to the wells and the reaction was stopped with phosphoric acid followed by absorbance measurements using a spectrophotometer.
RESULTS AND DISCUSSION
Paper I
Paper I deals with autoradiographic studies of 3H-vesamicol and 3H-QNB on cervical spinal cord sections from 11 patients with ALS and from four control cases. The investigation reports a trend towards increased binding of 3H-vesamicol in the ventral horn motor neuron area in ALS spinal cord sections as compared to control subjects (Fig. 3). This lack of decrease in expression of the ACh transporter, 3H-vesamicol, despite a profound loss of motor neurons in this particular area could be interpreted as an increased demand of synaptic vesicle formation in remaining motor neurons. It is known that motor neurons undergo a spatio-temporal denervation- reinnevration process during the general denervation in ALS, and it is postulated that remaining motor neurons sprout in a collateral manner, nurturing adjacent muscle fibers in order to prevent their death. It is reasonable to believe that the unaltered 3H-vesamicol binding in ALS spinal cord tissue reflects an increased activity in the few large remaining motor neurons.
Figure 3. Density of 3H-QNB and 3H-vesamicol binding sites in the motor neuron area of the ventral horn within ALS and control cervical spinal cord sections.
0 20 40 60 80 100 120 140 160 180
fmol/mg wet weight
3H-QNB 3H-vesamicol
ALS Controls
*
Paper II
Paper II describes levels of the antiapoptotic protein Bcl-2 and the preapoptotic protein Bax and the level of the caspase family member ICH- 1L in thoracic spinal cord sections from five ALS patients and five controls.
The study revealed unaltered levels of ICH-1 L and Bcl-2 in ALS compared to controls whereas Bax expression was up reglated in ALS cases. Our results are in accordance with a previous report, describing unaltered levels of Bcl-2 protein and increased bax mRNA levels in ALS spinal cord (Mu et al., 1996). We also performed DNA fragmentation analysis and found a substantial degree of DNA fragmentation indicating apoptosis in ALS motor neurons. These results are also supported by other groups (Migheli et al., 1994; Troost et al., 1995).
Paper III
Paper III demonstrates the mRNA levels of the neurotrophic factors GDNF and BDNF in muscle biopsies from five ALS patients, six patients with other neuromuscular diseases and eight healthy controls. We found increased levels of GDNF mRNA in the ALS group compared to the control groups (Fig. 4). This is in accordance with other reports (Lie and Weis, 1998; Yamamoto et al., 1999). The discrepancy between our results and the results presented by Yamamoto and co-workers in 1996 might be explained by different study design (Yamamoto et al., 1996). Yamamoto and colleagues studied GDNF expression in post-mortem muscle tissue from patients dying of the disease, whereas our study was based on muscle biopsy material obtained at a relatively early stage of the disease. It is possible that the level of GDNF initially is upregulated in degenerative conditions such as ALS but with time returns to normal levels.
BDNF levels were within the same range in the three groups analyzed in our investigation.
Paper IV
Paper IV describes the protein levels of the neurotrophic factors GDNF and BDNF in cerebrospinal fluid from 15 ALS patients and 11 healthy controls.
We found increased levels of GDNF protein in 12 out of 15 ALS patients compared to the controls where GDNF was not detectable (Fig. 4). BDNF was equally expressed in both groups studied. These results are in accordance with our previous findings in muscle biopsy tissue from ALS
patients (Paper III). These specific results of a marked upregulation of endogenous GDNF and GDNF mRNA in ALS CSF and muscle, respectively, are of special interest in relation to clinical trials where GDNF is administered to this group of patients.
Figure 4. GDNF expression in ALS and controls (ND = not detectable).
0 50 100 150 200
protein in CSF
(pg/ml)
mRNA in muscle
(arbitrary units)
ALS CONTROLS ALS CONTROLS
ND
CONCLUSION
This study has shown:
unaltered 3H-vesamicol binding in the ventral motor neuron area in ALS spinal cord in comparison to controls. The unaltered expression of 3H- vesamicol is detected despite a profound loss of motor neurons in the ALS cases. It is possible that 3H-vesamicol has the potential to act as a marker of metabolic activity in the remaining motor neurons in ALS (Paper I).
an upregulation of the proapoptotic protein Bax in motor neurons in ALS and DNA fragmentation in ALS spinal cord sections, both indicative of an ongoing apoptotic process (Paper II).
increased mRNA levels of the neurotrophic factor GDNF in muscle biopsies from patients with ALS. BDNF mRNA levels were found to be unaltered compared to controls (Paper III).
measurable levels of the neurotrophic factor GDNF in cerebrospinal fluid from patients with ALS in comparison to controls where GDNF was not detectable. Levels of BDNF were in the same range in both ALS patients and control subjects (Paper IV).
FUTURE PROSPECTS
From the perspective of the author future research should include an extended study to reveal in detail changes occuring in GDNF and BDNF levels in muscle and nerve tissue as a function of time in the state of degeneration. Such studies would most likely be performed in an animal model resembling the process of ALS. We have developed an experimental model in the rat, where it is possible to study both degeneration and regeneration by means of unilateral freezing of the sciatic nerve. Collection of distal muscle tissue, spinal cord and proximal and distal nerve samples at different time points after surgery will facilitate for a careful observation of changes in the levels of these neurotrophic factors during the denervation- reinnervation process. Such data will provide a better understanding of the process before making further extensive clinical trials in this group of patients.
Other factors of the GDNF family, such as persephin, artemin and neurturin, should also be further investigated in relation to neurodegeneration and ALS.
ACKNOWLEDGMENTS
This work has been carried out at the Department of Neuroscience, Neurology, University Hospital, Uppsala, supported by grants from the Swedish Medical Research Council, the Foundation of H. M. King Gustav V and H. M. Queen Victoria, the Swedish Association of Neurologically Disabled, the Swedish Natural Science Research Council, Gun and Bengt Björklund Foundation for ALS-research and the Swedish Cancer Foundation.
I wish to express my sincere gratitude to all of you who helped and supported me in this work and, especially:
Sten-Magnus Aquilonius, my supervisor, for sharing your great knowledge in the field of neurology and for giving me the opportunity to work on this exciting project.
Håkan Askmark for support, encouragement and constructive collaboration and for bringing new drive and energy into the project when you returned to Uppsala and rejoined the ALS research group.
Per-Göran Gillberg for enthusiasm and support during my first year as a Ph.D. student. Have any technical setbacks ever stopped you in the lab?
Also thanks for great pep talks and good and sincere advice.
Ted Ebendal and his research group for introducing me to the exciting field of neuroscience and for fruitful collaboration.
Dan Lindholm for interesting collaboration and encouragement.
Titti Ekegren for exchange of thoughts and ideas in the lab and for being a good friend and our many discussions about all and nothing.
Cecilia Gomes-Trolin for being such a generous and caring person and for excellent and thoughtful advice.
Co authors: Jonas Lindeberg, Ingela Nygren, Anders Johansson and Kaj Blennow for rewarding collaboration.
Gun Schönnings for invaluable help, always taking care of any administrative issue in no time at all.
friends and colleagues at ”Forskningsetapp III”, especially Inger Trångteg, Department of Developmental Neuroscience, especially Laura Korhonen and Susanne Hamnér, the Neurology ward 85D and the ALS team, especially Agneta Gustavsson, the staff at Autopsy and all the ALS patients and healthy volunteers who contributed to this thesis.
Anna Nordmark for great friendship in and out of the lab. Life isn’t the same without our daily chats over a cup of tea.
Niklas Finnström, my office mate at lab, for too many things to mention.
Thanks for being a good friend and for spoiling me with breakfast and numerous daily tea breaks in the lab as well as nice dinners. We’ve shared loads of laughs (especially while paragliding). Still waiting for the perfect late evening in the lab, watching ”Nattvakten” with you. Do you remember
”filippin”? You still owe me! Daniel for being a cute happy fellow. Thanks for helping me out with my dogs at times.
Patricia Lindgren for dear, genuine and sincere friendship and for always taking time for me, for so much fun and laughs and for discussions about everything under the sun, for being a great travel companion and for all pasta dinners we’ve shared while creating ”the scoring list”. Some day we’ll make that trip to New York again! Oliver, lets roll the die!
Patrick Doherty, my ”favourite Irish friend”, for care, support and wisdom and also for sharing my great interest in tea and English Springer Spaniels.
Marie Bengtsson for being such a spontaneous and generous friend and for all the fun we’ve had while practicing and ”performing” on violins and piano together.
Ylva Hedene for sincere and genuine friendship. Thanks for numerous advice (too much to mention) and extended phone conversations. You certainly know how to put things in perspective. Also thanks for all our talks over too many cups of tea, discussing the world of dogs, especially English Springer Spaniels, pedigrees and other details of breeding. Anders
for being an equally wonderful person. Also thanks for taking Saga in as a new member of your family.
Peter Heerdt for care and support and for always ”being on call”, helping me on a short notice with practical computer emergencies as well as for sharing my great interest in English Springer Spaniels. Monica and Richard for genuine care and support.
my wonderful extended family of aunts, uncles and cousins and their families for always making me feel right at home whenever we’ve met.
Thanks for all memorable get-togethers we’ve had over the years, whether on the ski slope or around the dinner table. May our get-togethers continue with coming generations.
my grandmother Ester, for deep, genuine interest and concern and for always keeping track on your children and all grandchildren and great grandchildren. Your 100th birthday party last year was the nicest (and only) one I’ve ever been to. Well done!
my two wonderful English Springer Spaniels:
Stina, whom I have so much to thank for. You’ve been a wonderful and loyal companion over the past six years, always greeting me smiling, with a wagging tail and a retrieved shoe, and you’ve always made sure I got some fresh air and exercise every day. You’ve been a great company at every meal, always hoping for a little treat. I’d also like to thank you for sharing the experience of being a mom and your puppies with me, 13 altogether.
Thea, my baby pup, for the nicest of dispositions and for being the cutest pup I’ve ever laid eyes on. The alarm clock has been stored away in the closet now, together with any shoes you were kind enough to leave unchewed.
My life would have been less enjoyable, probably even miserable without:
quince tea and Arla, ice cream, annual Christmas celebrations, Q Image, Frédéric Chopin (especially the ”minor” compositions), ”turkisk peppar”, Birkenstock, ICA-hörnan, poppy seed bread, Eudora and Outlook Express, Friskis & Svettis, the hilarious mind behind question #8, who blessed me with so much laughs while finalizing the thesis, Björn Ehlin and Therese
Angserud, Tele2 (you instantly made me a Gold-member!), the Sauna Association, ”Springerklubben” and all my ESS and breeder friends, Trolltyg in Uppsala, my dear piano, Frans, Pepsi, Ludwig, Tina, Peggy, Julia, Alice, Elvis, Simon, Rocky, Poppe, Fanny and Saga with families,
”Brunkullans blandning”, ”lussebullar”, the extended airline network, my red ”Peak” jacket and the change of seasons.
Finally I’d like to thank the people closest to my heart:
my brothers Lars and Per for love, support and care and for being such great friends. We’ve had so much fun together! Lina for being like a sister to me.
my parents Ruth and Sören, for endless love, support and encouragement and for always trusting and believing in me no matter how wild the ideas I might have had over the years and for always being there for me, helping me with practical things in life.
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