Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 4 uppsatser.

Cognitive impairment in amyotrophic lateral sclerosis (ALS) is characterized by deficits on tests of executive functions however the contribution of processing speed is unknown. By contrast, multiple sclerosis (MS) is a disorder in which slowed processing speed is regarded as the core deficit, however, methodology is often confounded by tasks which depend on motor speed. MRI studies have revealed multi-system cerebral involvement in ALS, with evidence of reduced white matter volume and integrity in predominantly frontotemporal regions. The current study had two aims. Firstly, to investigate whether cognitive impairments in ALS and MS are due to executive dysfunction or slowed processing speed, independent of motor dysfunction. Secondly, to investigate the relationship between specific cognitive impairments and the integrity of distinct white matter tracts in ALS. Twenty-nine ALS patients, twenty-five MS patients, and matched healthy control groups were administered a dual task paradigm and processing speed tasks in which stimulus presentation times were manipulated. In addition background measures of executive functioning, working memory, verbal memory, and language were administered. White matter integrity was investigated using region-of-interest (ROI) and tract based spatial statistics (TBSS) analyses of diffusion MRI data. ALS patients did not show impairments in tests of processing speed, but deficits were revealed in the dual task, as well as background tests of executive functioning, working memory, and verbal memory. MS patients also exhibited deficits in the dual task as well as background tests of executive functioning, working memory, and verbal memory. However, in contrast to ALS patients, a processing speed deficit was also observed in MS. ROI analyses revealed significant differences in fractional anisotropy (FA) and mean diffusivity (<D>) between ALS patients and healthy controls. Reduced integrity was observed in the corticospinal tracts and prefrontal and temporal white matter tracts including uncinate fasciculus, inferior longitudinal fasciculus, and regions of the cingulum. Significant differences also emerged in the white matter underlying the superior, medial and inferior frontal gyri, and the temporal gyri. Similar group differences were found in the TBSS analyses; ALS patients displayed prominent changes in the corticospinal tract and corpus callosum as well as extensive changes in prefrontal and temporal tracts and association fibres. Correlations between task performance and ROI parameters revealed that dual task performance was associated with FA in the middle frontal gyrus white matter while letter fluency indices correlated with FA in the corpus callosum and corticospinal tracts. Furthermore, verbal memory performance correlated with FA in the inferior longitudinal fasciculus and working memory performance correlated with <D> in uncinate fasciculus and hippocampal portion of the cingulum. Correlations with TBSS revealed significant associations between letter fluency indices and FA in the corticospinal tracts and anterior corpus callosum. The current study demonstrates that cognitive impairment in ALS is not due to slowed processing speed. Moreover dual task deficits are related to distinct prefrontal tract involvement in ALS, whilst fluency deficits may reflect decreasing callosal integrity. Deficits in working memory and verbal memory are related to white matter changes in fibre bundles connecting prefrontal, temporal, and limbic structures.

Amyotrophic Lateral Sclerosis is the most common type of motor neuron disease, characterized by progressive degeneration of the upper and lower motor neurons. Sufferers present with symptoms of muscle weakness and this quickly develops on to paralysis and finally death due to respiratory failure within 5 years of disease onset. Although the majority of cases are sporadic, about 10% are familial and it is hoped that through the investigation of these few cases a greater understanding of the disease process, the reasons for its delayed onset and vulnerability of motor neurons will be achieved. Recently a novel mutation linked to ALS was discovered in an evolutionary conserved protein named Vesicle associated membrane protein (VAMP) associated protein B (VAPB). VAPB is an integral type II membrane protein localised at the Endoplasmic Reticulum and thought to have a role in protein transport. The orthologue in Drosophila has been shown to be involved in the homeostatic regulation of bouton formation at the Neuromuscular Junction through an association with the microtubule network. To elucidate the mechanism through which this protein causes ALS, Pennetta et al have created a Drosophila model of the disease by expressing the mutated orthologue in the fly. To complement this model, I have undertaken a number of biochemical experiments to look for potential interactors of the VAP proteins. The yeast two hybrid system utilises the yeast GAL4 transcriptional activator to indicate a protein interaction within a yeast cell and can be used to test a cDNA library for interactors. Through this technique a number of interesting binding partners have been found that may play crucial roles in the progression of the disease.

Bakgrund: Amyotrofisk lateralskleros (ALS) tillhör gruppen motorneuronsjukdomar. Det är en obotlig neurologisk sjukdom som drabbar cirka 200 personer per år i Sverige. De viljestyrda musklerna i kroppen förtvinar vilket leder till att personen får problem med tal och andning. Sjukdomsförloppet varierar beroende på vilken form av motorneuronsjukdom personen drabbas av, vid ALS avlider personen oftast inom 5 år. Syfte: Syftet med litteraturstudien var att beskriva personers erfarenheter av att leva med ALS samt beskriva de inkluderade vetenskapliga artiklarnas undersökningsgrupp. Metod: En beskrivande litteraturstudie som består av 11 kvalitativa artiklar. Huvudresultat: Några patienter var missnöjda med sjukhusvården, de möttes av okunnig personal och blev skickade mellan olika vårdenheter innan diagnosen kunde fastställas. Det ledde till att patienterna själva fick söka efter svar. Patienterna beskrev flera negativa känslor med diagnosen ALS. Under sjukdomsförloppet uppstod även positiva känslor som gjorde att patienterna fortsatte att kämpa. Patienterna använde sig av olika strategier som existentiella tankar, acceptans för hjälpmedel och att ta hjälp av en utomstående vårdare. Stöd från familj och vänner var betydelsefulla för att uthärda sjukdomen och den förväntade döden. Slutsatser: Personer som drabbats av ALS upplevde en känslomässig bergochdalbana, som pendlade mellan positiva och negativa känslor. För flera patienter var det viktigt att ta en dag i taget för att finna meningsfullhet. Sjukdomsupplevelsen är individuell vid ALS och det är viktigt att sjuksköterskan använder sig av personcentrerad vård. Det är betydelsefullt att sjuksköterskan tar till sig patienters och anhörigas erfarenheter för att bevara dess integritet och autonomi. / Background: Amyotrophic lateral sclerosis (ALS) belongs to a group of motor neuron diseases. It is an incurable neurological disease that affects approximately 200 people per year in Sweden. The involuntary muscles of the body atrophies which causing the person problems to speak and breath. The disease progression varies depending on the version of motor neuron disease the person suffering, at ALS person usually dies within 5 years. Aim: The purpose of this study was to describe people's experiences of living with ALS and describe the study group of the included scientific articles. Method: A descriptive literature study consisting of 11 qualitative articles. Main Results: Some patients were not satisfied with the hospital care, they were met by ignorant staff and were sent between different care units before a diagnosis could be decided. At the end the patients themselves were searching for answers themselves. Patients describe several negative feelings diagnosed with ALS. During the progression of the disease even positive emotions came and encouraged the patients continue to struggle. Patients used different strategies like existential thoughts, acceptance of aid and help from outside caregiver. Support from family and friends were important to cope with the disease and the expected death. Conclusions: People who suffer from ALS experienced an emotional roller coaster, which commuted between positive and negative feelings. For many patients, it was important to take one day at a time to find meaningfulness. The experience from the illness is individual from ALS and it is important that the nurse uses the person-centered care. It is important that the nurse learn from the patients and relatives experience to preserve the integrity and autonomy.

The spinal cord is a complex structure responsible for processing sensory inputs and motor outputs. As such, the developmental and spatial organization of cells is highly organized. Diseases affecting the spinal cord, such as Amyotrophic Lateral Sclerosis (ALS), result in the disruption of normal cellular function and intercellular interactions, culminating in neurodegeneration. Deciphering disease mechanisms requires a fundamental understanding of both the normal development of cells within the spinal cord as well as the homeostatic environment that allows for proper function. Biological processes such as cellular differentiation, maturation, and disease progression proceed in an asynchronous and cell type-specific manner. Until recently, bulk measurements of a mixed population of cells have been key in understanding these events. However, bulk measurements can obscure the molecular mechanisms involved in branched or coinciding processes, such as differential transcriptional responses occurring between subpopulations of cells. Measurements in individual cells have largely been restricted to 4 color immunofluorescence assays, which provide a solid but limited view of molecular-level changes. Recently, developments in single cell RNA-sequencing (scRNA-Seq) have provided an avenue of accurately profiling the RNA expression levels of thousands of genes concomitantly in an individual cell. With this increased experimental precision comes the ability to explore pathways that are differentially activated in subpopulations of cells, and to determine the transcriptional programs that underlie complex biological processes. In this dissertation, I will first review the key features of scRNA-Seq and downstream analysis. I will then discuss two applications of scRNA-seq: 1) the in vitro differentiation of mouse embryonic stem cells into motor neurons, and 2) the effect of the ALS-associated gene SOD1G93A expression on cultured motor neurons in a cellular model of ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neurological disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have revealed that many ALS-associated genes are involved in autophagy, but the role of this pathway in motor neurons remains poorly understood. Here, we use the SOD1G93A mouse model to investigate the role of autophagy in ALS. We find neuronal subtype-specific regulation of autophagy over the course of disease progression. Vulnerable motor neurons form large GABARAPL1-positive autophagosomes that engulf ubiquitinated cargo recognized by the selective autophagy receptor p62. Other motor neurons and interneurons do not engulf cargo within GABARAPL1-positive autophagosomes and instead accumulate somatodendritic aggregates. To investigate whether motor neuron autophagy is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 is specifically disrupted in motor neurons. Phenotypic analysis of these mice revealed that autophagy is dispensable for motor neuron survival but plays a key role in regulating presynaptic structure and function. By crossing these mice to the SOD1G93A mouse model, we find that autophagy inhibition accelerates early neuromuscular denervation and neurological dysfunction. However, loss of autophagy in motor neurons eventually leads to an extension of lifespan, and this is associated with reduced pathology in interneurons and glial cells. These data suggest that vulnerable motor neurons rely on autophagy to maintain neuromuscular innervation early in disease. However, autophagy eventually acts in a non-cell autonomous manner to promote disease spread and neuroinflammation. Our results reveal counteracting roles for motor neuron autophagy early and late in ALS disease progression.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two devastating neurodegenerative diseases in urgent need of therapeutic intervention. The last seven years has been a period of great progress in understanding these disorders separately and as a disease spectrum. Most notable is the discovery of the hexanucleotide GGGGCC expansion in the C9ORF72 (C9) gene, which is the greatest known cause of inherited and sporadic forms of these two diseases. In response to this groundbreaking discovery, we set out to elucidate the molecular mechanisms of C9 pathogenesis with a focus on the expanded RNA transcripts derived from the C9 expansion. Our two primary goals have been to contribute to the worldwide efforts to understand the primary toxic insults of this mutation that will ultimately shape therapeutic development, and to identify molecular criteria that can be used to define new links between these diseases and undetermined genetic factors. In the introduction, we review the broad conceptual links between RNA binding proteins (RBPs), mRNA regulation, and neurodegeneration. This review contains substantial discussion of ALS, FTD, and C9, as well as related neurodegenerative, neuromuscular and repeat expansion diseases. In addition to providing a detailed history of molecular mechanisms proposed for these disorders, this section serves as a justification for our focus on the C9 RNA, RBP sequestration, and altered splicing that we describe in the following chapters. Chapter two consists of our 2016 Elife paper on sequestration of the RBP hnRNP H and resulting splicing changes in C9ALS-FTD afflicted individuals. In this paper, we sought to identify the most biochemically sound candidate for the proposed RBP sequestration hypothesis. We found that the splicing factor hnRNP H binds with high affinity to the repeat sequence and likely has a role in regulating the transition of the repeat RNA from linear to G-quadruplex (G-Q) conformation. Importantly, we identified functional deficiency of this protein in patient brains, as evidenced by dysregulation of known hnRNP H splicing targets, and loss of soluble hnRNP H. Chapter three consists of recently submitted work on the molecular links between C9ALS/FTD, and sporadic ALS/FTD at large. Building upon our findings in C9ALS-FTD, we have sought to ask whether the changes to hnRNP H we predicted would occur in C9 expansion carriers as a result of the repeat RNA might also occur independent of this expansion. We found that indeed half of all patients in a cohort of 50 sporadic ALS, ALS-FTD, and FTD brains demonstrated hnRNP H sequestration and accompanying splicing changes, a pattern we refer to as like-C9. Like-C9 patients may be thought of as phenocopies of C9 expansion carriers, in that they not only present with similar clinical symptoms, but also possess remarkably similar molecular signatures of RBP dysfunction. While the genomic origins of like-C9 remain unknown, we propose that they are suggestive of repeat expansions analogous to C9, much like what is seen in DM1 and DM2, and HD and HDL2 (discussed in Ch. 1). This work has provided the foundation for our ongoing search for novel genomic expansions that confer increased susceptibility to ALS/FTD.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset paralytic disorder for which there is currently no cure. Underlying the disease mechanism of ALS is the spontaneous pathologic degeneration of motor neurons (MNs). Understanding the molecular mechanisms underlying spontaneous and selective MN demise is critical to the development of rational therapeutic strategies. In the current work, utilizing established in vitro models of ALS, I demonstrate that necroptosis, a form of caspase-independent programmed cell death (PCD), drives MN death. Pharmacologic inhibition and/or genetic silencing of receptor interacting protein kinase-1 (RIPK1), receptor interacting protein kinase-3 (RIPK3), and mixed lineage kinase domain-like-protein (MLKL) rescued MN death in vitro. While this core machinery was conserved, the requirement of nuclear factor kappa-B (NF-κB) and Bcl-2-associated X protein (Bax) deviated from known models of necroptosis. This divergence led me to consider that there may be a MN-specific program of necroptosis. Thus, I then used unbiased approaches, by meta-analyzing a gene expression signature captured from MNs undergoing cell death in vitro, to explore MN cell death drivers that may be engaged upstream or downstream to RIPK1/RIPK3/MLKL. I also explored the relevance of necroptosis to MN disease in vivo, in part by deleting RIPK3 from a genetic mouse model of familial ALS. Overall this approach did not rescue motor neuron loss, and there was no improvement in motor function, disease onset, or survival in these animals. I conclude that while necroptosis machinery drives motor neuron death in in vitro models of ALS, more work needs to be done to (1) assess the motor neuron-specific cell death program, and (2) evaluate the relationship, if any, of necroptosis to motor neuron disease in vivo.

Amyotrophic Lateral Sclerosis (ALS) is a rapid and fatal neurodegenerative disease marked by progressive muscle weakness and wasting. Approximately 50% of people with ALS experience changes in cognition and behaviour. Previous research has been mixed as to whether cognition declines over the course of ALS, or whether it is related to proxies of disease progression (e.g., functional disability scales). However, this research has suffered from limitations including the use of inappropriate measures of cognition, imprecise measures of disease progression, high attrition, practice effects, and biased analytic approaches. Fortunately, recent advances in clinical assessment have provided accurate measures of neuropsychological functioning and disease progression, namely, the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and the King's Clinical Disease Staging. The present study aims to utilise recent advances in ALS disease metrics to overcome previous limitations and explore the evolution of cognitive and behavioural dysfunction over the course of ALS. Specifically, the aims of the present project are to 1) develop alternate forms of the ECAS to accommodate repeated longitudinal assessment; 2) examine how cognition and behaviour relate to clinical disease stages in ALS; 3) evaluate how cognitive and behavioural symptoms evolve over the course of the disease in ALS; and 4) explore clinicians' attitudes toward cognitive and behavioural screening in ALS. To achieve Aim 1, two new versions of the ECAS (ECAS-B and ECAS-C) were developed and administered to a group of age, education, and gender matched controls to that of the original ECAS-A validation study. Results demonstrate that the alternate forms of the ECAS (B and C) were equivalent to the original ECAS-A, reducing practice effects and possessing excellent inter-rater reliability. The ECAS forms were administered longitudinally to a separate group of healthy controls. Over an interval of 4 months, the ECAS-A-B-C showed no evidence of practice effects and excellent test-retest reliability validating their utility in the longitudinal monitoring of cognition in ALS. The ECAS forms were then used in an international multi-centre clinical sample of 161 ALS patients and 80 matched controls to achieve Aim 2. Patients were grouped into their King's Clinical Disease Stage at time of testing. Analysis revealed a significant cross-sectional relationship between disease stage and ALS-Specific cognitive functions, driven by a decline in verbal fluency performance. A significant relationship was also observed between disease stage and behavioural features. By end-stage disease 80% of patients demonstrated neuropsychological impairment. Participants were followed up longitudinally to explore the progression of cognitive and behavioural symptoms. Latent Growth Curve models of the ECAS subdomains (utilising the alternate versions) demonstrated a significant decline in ALS Specific cognitive, but not behavioural, functioning over time. This decline was explained by advancing disease stage, the presence of the C9orf72 repeat expansion, and years of education. Rate of change in ALS Non-Specific functions was dependent on baseline performance. Visuospatial functions and perseveration declined at similar rates and were distinct from language, fluency, apathy, and disinhibited behaviour. Cluster analysis of patients revealed a three-cluster solution with one group demonstrating no significant decline, a second group with mild cognitive and behavioural decline, and a third group with more severe neuropsychological decline. When data was restructured by diseases stage, rather than time, longitudinal results were similar to cross-sectional findings. To examine clinician's attitudes to cognitive and behavioural screening. Fourteen Health Care Professionals (HCP) working in ALS (Neurologists, Psychologists, and Clinical Care Specialists) were interviewed. Thematic analysis revealed that HCPs recognised the importance of cognitive and behavioural screening in ALS, but that it is not common practice. Important barriers to screening were reported including other members of staff, a lack of resources, and issues concerning patients and their families. Participants suggested increasing training and psychology input, and making screening a standardised protocol to all patients may alleviate these barriers. Cognition and behaviour are critically related to advancing disease stage, both cross-sectionally and longitudinally. Declining cognitive and behavioural symptoms has important implications for clinical practice, caregiver impact, and end-of-life decision making. However, clinicians report that cognitive and behavioural screening is not common practice and that significant barriers exist. The newly developed alternate forms of the ECAS provide an accurate, effective, and clinically useful means of monitoring cognitive function over the course of the disease in ALS.

This thesis examines the pathophysiology of motor neurone dysfunction, along with site of disease onset, in amyotrophic lateral sclerosis (ALS). The rationale for this thesis is the "dying forward" hypothesis, which suggests that corticomotoneurons cause anterograde excitotoxic degeneration of motor neurons in ALS. Initially, axonal excitability studies were applied to ALS patients and revealed widespread axonal ion channel dysfunction, with increases in persistent Na+ conductances and reduction in K+ currents. Such changes result in axonal hyperexcitability, thereby resulting in generation of fasciculations and cramps. Subsequently, axonal excitability studies were applied to Kennedy's disease (KD) patients, a pathological control group, revealing similar changes to ALS and suggesting that upregulation of persistent Na+ conductances was responsible for generation of fasciculations. To better understand the mechanisms underlying fatigability and to assess whether Na+/K+ pump dysfunction contributes to neurodegeneration in ALS, activity-dependent changes in axonal excitability were measured after a maximal voluntary contraction. The increase in threshold was more pronounced in ALS patients with predominantly lower motor neuron involvement, suggesting that peripheral factors were responsible for fatigue in ALS and that Na+/K+ pump function was preserved. Having documented abnormalities of axonal excitability, a novel threshold tracking transcranial magnetic stimulation (TMS) technique was developed for assessment of cortical excitability. This technique overcomes the marked variability in the motor evoked potential with consecutive stimuli, a major limitation of the previous "constant stimulus" technique. After establishing normative data, threshold tracking TMS established that cortical hyperexcitability was an early and prominent feature in ALS. Similar changes were found in flail-arm variant ALS, a pure lower motor neuron form of ALS. In KD patients, cortical excitability was normal, thereby suggesting that cortical hyperexcitability is a primary event in ALS rather than a down-regulation of inhibitory control over the motor cortex in order to compensate for anterior horn cell loss. In order to determine whether cortical hyperexcitability underlies motor neurodegeneration, longitudinal studies were undertaken in familial ALS subjects with the copper/zinc superoxide-dismutase-1 gene mutation. These studies established that cortical hyperexcitability precedes the development of clinical ALS, thereby suggesting that cortical hyperexcitability underlies the basis of motor neurodegeneration in familial ALS.