Dissertations / Theses on the topic 'Amyotrophic lateral sclerosis'

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised clinically by rapidly progressive paralysis leading ultimately to death from respiratory failure. There is no cure for ALS and no definitive explanation for the onset and rapid progression of motor neuron degeneration. Genetics is a known risk factor for a portion of familial cases. However, the role of genetics in the commoner sporadic form of the disease is poorly understood, although numerous genes have been implicated. The primary aim of this thesis project is to uncover the genetic causes that underlie ALS. To accomplish this goal, the main focus of this thesis is to perform genome-wide association analysis of sporadic ALS using high density SNP arrays. This thesis describes the first and the largest genome-wide association studies of ALS to date. Results demonstrate that there is no single large effect susceptibility variant underlying a large proportion of ALS, such as ApoE in Alzheimer’s disease. However, the genotyping data has been made publically available and the digital nature of this data means that it is a resource that can grow with future studies. Beyond genome-wide association, this thesis describes work using linkage, haplotype and sequence analysis to investigate the genetic overlap between ALS and frontotemporal dementia. Lastly, this thesis presents a novel method for linkage analysis using high throughput SNP arrays. Ultimately, it is hoped that by uncovering the genes that cause ALS, such knowledge will shed light on the pathogenic mechanisms underlying motor neuron degeneration and potentially lead to new rational therapies effective in slowing or even halting disease progression.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease of motor neurons characterized by muscle paralysis and death within 3-5 years of onset. However, due to unknown mechanisms, the extraocular muscles (EOMs) remain remarkably unaffected. The EOMs are highly specialized muscles that differ from other muscles in many respects, including innervation and satellite cells (SCs). Understanding whether these factors play a role in the relative sparing of EOMs in ALS could provide useful clues on how to slow down the progression of ALS in other muscles. The EOMs and limb muscles from terminal ALS patients and age-matched controls as well as the commonly used SOD1G93A ALS mouse model were studied with immunofluorescence. Antibodies against neurofilament and synaptophysin were used to identify nerves and neuromuscular junctions (NMJs); against Pax7, NCAM, MyoD, myogenin, Ki-67, dystrophin and laminin, to identify SCs and their progeny in EOMs and limb muscles. The proportion and fiber size of myofibers containing myosin heavy chain (MyHC) slow tonic and MyHC slow twitch were also determined in human EOMs. The abundance of SCs differed extensively along the length of control human EOMs, being twice as abundant in the anterior portion. Pax7-positive cells were also detected in non-traditional SC positions. EOMs from terminal ALS patients showed similar numbers of resting and activated SCs as the controls. In limb muscles of ALS patients, the number of resting and activated SCs ranged from low (similar to normal aged, sedentary individuals) to high numbers, especially in muscles with long duration of disease and varied between the upper and lower limbs. The EOMs maintained a high degree of innervation compared to hindlimb muscles of symptomatic SOD1G93A mice. MyHC slow tonic fibers were less abundant in ALS patients than in controls. The change seemed more pronounced in bulbar onset patients, and in this group of subjects only, there was a strong association between decline in MyHC slow tonic fibers and age of death. Notably, the decline in MyHC slow tonic fibers was unrelated to disease duration. Our data suggested that SCs play a minor role in the progression of ALS in general and in the sparing of the EOMs in particular. The generally preserved innervation in the EOMs of G93A mice may reflect distinct intrinsic properties relevant for sparing of the oculomotor system.  Even though the EOMs are relatively spared in ALS, MyHC slow tonic myofibers were selectively affected and this may reflect differences in innervation, as these fibers are multiply innervated.
Amyotrofisk lateralskleros (ALS) är en obotlig neurodegenerativ sjukdom som främst påverkar kroppens viljestyrda motoriska nervceller. ALS leder till förlamning, muskelförtvining och slutligen döden genom andningssvikt, vanligen inom tre till fem år efter sjukdomsdebuten. Av okända anledningar så bibehålls ögonmusklernas funktion mycket bättre vid ALS i jämförelse med andra muskler och är hos merparten av patienter i stort sett opåverkade. Ögonmusklerna är mycket specialiserade muskler som skiljer sig från andra muskler i kroppen på flera sätt, bland annat genom deras unika nervförsörjning och genom de satellitceller – muskelspecifika stamceller, som finns i dem. En ökad förståelse för hur dessa faktorer inverkar på ögonmusklernas motståndskraft vid ALS skulle kunna ge värdefulla ledtrådar till hur man skulle kunna sakta ned sjukdomens fortskridande i andra muskler vid ALS. Ögonmuskler och extremitetsmuskler från avlidna ALS-patienter och åldersmatchade friska kontroller, tillsammans med transgena möss med den sjukdomsalstrande mutationen SOD1G93A, studerades genom immunfluorescens och efterföljande mikroskopering. Antikroppar mot molekylerna Pax7, NCAM, MyoD, myogenin, Ki-67, laminin och dystrofin användes för att identifiera satellitceller och deras dotterceller i ögonmuskler och extremitetsmuskler. Antikroppar mot neurofilament och synaptofysin användes för att identifiera nerver och neuromuskulära synapser hos transgena SOD1-möss. Antikroppar mot toniska (tonic) och ryckande (twitch) muskelmyosinkedjor användes för att bestämma proportionen av och storleken på dessa typer av muskelfibrer i ögonmuskler från avlidna ALS-patienter och friska kontroller. Mängden satellitceller varierade mellan de främre och de mer bakre delarna i friska, humana ögonmuskler och var dubbelt så många i den främre delen av muskeln jämfört med den mellersta och bakre delen av muskeln. Celler som uttryckte satellitcellsmarkören Pax7 hittades även i icke-traditionella satellitcellspositioner i ögonmusklerna. Mängden satellitceller i ögonmusklerna från ALS-patienter var samma som hos friska kontroller. I extremitetsmusklerna hos ALS-patienter varierade mängden satellitceller mellan låga nivåer (liknande de hos friska åldrade, inaktiva individer) till höga nivåer, särskilt i muskler där sjukdomen fortskridit under lång tid. Dessutom varierade mängden satellitceller mellan övre och nedre extremiteter. Hos symptomatiska SOD1G93A-möss hade ögonmusklerna en mycket välbevarad innervation jämfört med bakbensmusklerna, där många neuromuskulära synapser saknade kontakt mellan nerven och motorändplattan. Proportionen muskelfibrer med toniska muskelmyosinkedjor var lägre hos ALS-patienter jämfört med friska kontroller. Denna minskning var tydligare hos patienter där sjukdomssymtomen hade debuterat i tugg- och ansiktsmuskulaturen – så kallad bulbär ALS. Dessutom fanns det i den här gruppen, men ingen annan studerad grupp, en stark korrelation mellan nedgången i toniska fibrer och patientens ålder. Värt att notera är att minskningen av toniska muskelfibrer saknade korrelation med hur länge patienten hade varit sjuk i ALS. Den generellt välbevarade innervationen i ögonmusklerna hos SOD1G93A-möss kan spegla distinkta inneboende egenskaper hos ögonmusklerna som är av vikt för bevarandet av ögonrörligheten vid ALS. Gällande satellitceller så antyder våra data att satellitceller och deras regenerativa kapacitet spelar en försumbar roll vid ALS i allmänhet och vid ögonmusklernas bevarande i synnerhet. Slutligen, även om ögonmuskler generellt är välbevarade vid ALS så är toniska muskelfibrer märkbart påverkade och detta kan spegla skillnader mellan olika nervcellsgruppers känslighet vid ALS.

Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive neurodegenerative disease, and is fatal within 3-5 years of onset. Metabolic dysfunctions have consistently been identified in ALS, although its role in pathogenesis remains unclear. In this thesis, I apply a metabolomic approach using 1H NMR spectroscopy and Gas Chromatography-Mass Spectrometry in a range of disease models of increasing biological complexity, as well as patient tissues, in order to reveal perturbations to the metabolic network that may impact the course of the disease. I examined alterations to metabolism in the motor neuron-like NSC-34 cell line, and found that mutant SOD1 led to increased glycolysis to divert glucose from oxidative metabolism, and a broad intracellular amino acid depletion. The contribution of non-cell autonomous processes were also investigated in an astrocyte-motor neuron co-culture model, where mutant SOD1 produced varying perturbations to glycolysis and oxidative stress responses in each cell type, together with decreased branched-chain amino acid catabolism and glutamine-glutamate production that may indicate impaired neurotransmitter recycling. I also found different metabolic responses to mutant SOD1 in two strains with varying rates of disease progression, suggesting a role for early metabolic responses to mutant SOD1 in affecting the course of disease. In addition, I identify a metabolic signature for C9ORF72 ALS in cerebellum tissue, providing evidence that the hexanucleotide repeat expansion leads to distinctive metabolic changes in the CNS. Overall, I demonstrate the applicability of metabolomics in ALS research, particularly in revealing hidden metabolic subtypes of the disease. This opens opportunities to improve our understanding of the processes leading to motor neuron death in ALS, and highlights the potential use of metabolomics as a tool to develop therapies targeted to the individual metabolic responses of individuals susceptible to ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive debilitating disease of the upper and lower motor neurons. Median survival of ALS patients is consistently estimated at between 2-3 years from symptom onset, with some evidence that survival is increasing due to improved care. There are few well-established risk factors for ALS, and there is conflicting evidence regarding the trends in ALS incidence and mortality over the past several decades. In Chapter I we investigate the trends in ALS incidence and mortality in Denmark between 1970 and 2009. We employed age-period-cohort models to model both the incidence and mortality rates of ALS over time for the first time. We found a significant rise in ALS incidence and mortality over several decades, and we observed evidence for a birth cohort component to the rise in ALS, which is consistent with an environmental cause of ALS. In Chapter II we investigate the role of physical trauma – both head and other – in the development of ALS. We employed the Danish registries and linked health data from the hospital system to prior diagnoses for physical trauma. We found a borderline significant association between physical trauma and ALS, which grew stronger upon restricting to physical traumas before the age of 55. Chapter III concerns the risk of ALS in those employed by the military in Denmark. We linked occupational records from the Danish Pension Fund to health records of the hospital system. We found a significantly elevated rate of ALS among those who had been previously employed by the military, with the highest rates in the decade immediately following cessation of employment. These analyses strengthen the knowledge base for the epidemiology of ALS, and suggest future avenues of research to further understand the etiology of the disease.

L’esclerosis lateral amiotròfica (ELA) és una malaltia neurodegenerativa devastadora, per la qual actualment no existeix cap tractament. L’ELA es caracteritza per la pèrdua progressiva de motoneurones (MN) tan superiors com inferiors i la consegüent atrofia muscular. A dia d’avui es desconeix el mecanisme molecular específic que promou la mort de les MN, però s’ha relacionat amb diferents processos que inclouen tant les MN com les cèl·lules del voltant com pot ser l’estrès oxidatiu, la inflamació o l’agregació de proteïnes com la superòxid dismutasa 1 (SOD1). En aquesta tesis nosaltres proposem dues estratègies terapèutiques per L’ELA: incrementar l’eficiència de silenciament de la proteïna mutada SOD1 mitjançant teràpia gènica i estudiar el rol de l’àcid lisofosfatídic (LPA) com a mediador de la inflamació, en la fisiopatologia de l’ELA. En el primer capítol, amb l’objectiu de reduir els nivells de RNA de la proteïna mutada SOD1, nosaltres administrem oligonucleòtids antisentit (ASO) conjugats a lligands específics per tal d’incrementar la seva internalització a les cèl·lules. Els resultats presentats revelen la eficient internalització d’aquesta teràpia a neurones i cèl·lules glials. A més, la conjugació del ASO amb el lligand DCPP redueix de manera més eficient els nivells de RNA de SOD1 que no pas els ASO no conjugats. Tot i això l’administració del ASO conjugat al lligand DCPP en els ratolins SOD1G93A, el model experimental de ALS, no presenten afectes terapèutics. Pel que fa el segon capítol, com que la inflamació és un tret comú de la majoria de les condicions neurològiques i sabent que el receptor LPA2 contribueix en la fisiopatologia de la lesió medul·lar, ens preguntem si l’LPA podria contribuir en la fisiopatologia de l’ELA. Per això vam creuar ratolins nuls pel receptor LPA2 amb ratolins SOD1G93A. Els resultats demostren que l’absència del receptor LPA2 retarda la progressió de la malaltia i evita l’atròfia muscular en ratolins SOD1G93A. Col·lectivament els resultats que presentem en aquesta tesis aporten nova informació que podria servir per desenvolupar estratègies pel tractament de l’ELA.
La esclerosis lateral amiotrófica (ELA) es una enfermedad neurodegenerativa devastadora, por la cual actualmente no existen ningún tratamiento. La ELA se caracteriza por la pérdida progresiva de motoneuronas (MN) superiores e inferiores y la consiguiente atrofia muscular. Hoy en día se desconocen los mecanismos moleculares específicos que promueven la muerte de estas MN, aunque se han relacionado con múltiples procesos que incluyen tanto las MN como las células subyacentes, tales como el estrés oxidativo, la inflamación o la agregación de proteínas como la superóxido dismutasa 1. En esta tesis nosotros proponemos dos estrategias terapéuticas para el ELA: incrementar la eficiencia del silenciamiento de la proteína mutada SOD1 mediante terapia génica y estudiar el rol del ácido lisofosfatídico (LPA) como mediador de la inflamación, en la fisiopatología del ELA. En el primer capítulo, con el objetivo de reducir los niveles de RNA de la proteína mutada SOD1, administramos oligonucleótidos antisentido (ASO) conjugados a ligandos específicos, por así incrementar su internalización a las células. Los resultados presentados revelan la eficiente internalización de esta terapia en neuronas y células gliales. Además, la conjugación del ASO con el ligando DCPP reduce de manera más eficiente que los ASO no conjugados, los niveles de RNA de SOD1 en los ratones SOD1G93A. Sin embargo, la administración del ASO conjugado con el ligando DCPP en los ratones SOD1G93A no presenta efectos terapéuticos. En el segundo capítulo, como la inflamación es un factor común en varias condiciones neurológicas y sabiendo que el receptor LPA2 contribuye en la fisiopatología de la lesión medular, nos preguntamos si el LPA podría contribuir en la fisiopatología del ELA. Por esto cruzamos ratones nulos por el receptor LPA2 con ratones transgénicos SOD1G93A. Nuestros resultados demuestran que la ausencia del receptor LPA2 retarda la progresión de la enfermedad y evita la atrofia muscular en ratones SOD1G93A. Colectivamente los resultados presentados en esta tesis aportan nueva información que podrían servir para desarrollar estrategias para el tratamiento de la ELA.
Amyotrophic lateral sclerosis (ALS) is a devasting neurodegenerative disorder with no effective treatment currently available. ALS is characterized by the progressive loss of both upper and lower motoneuron (MN) and the consequent muscle atrophy. Nowadays the specific molecular mechanism that underline the MN death is unknown, however has been related several dysfunction mechanisms in MNs and the surrounding cells, such: oxidative stress, inflammation or aggregation of aberrant proteins like superoxide dismutase 1 (SOD1). In this thesis we propose two therapeutic strategies for ALS: increase the silencing efficiency of the mutated SOD1, by means of gene therapy and study the role of the lysophosphatidic acid (LPA) in the pathophysiology of ALS. In the first chapter, with the aim to reduce the RNA levels of the SOD1 mutated, we administrated antisense oligonucleotide (ASO) conjugated to specific ligands to increase the internalization of the molecule. The results present here reveal the efficient internalization of the therapy in neurons and glia cells. Furthermore, the conjugated- ASO with the ligand DCPP reduce more efficiently than the unconjugated ASO, the SOD1 RNA levels in the SOD1G93A mice, an experimental model of ALS. However, the administration of the conjugated-ASO with the ligand DCPP in the SOD1G93A mice, does not present therapeutic effects. In the second chapter, since inflammation is a hallmark of most neurological conditions and LPA2 has been reported that contribute to the pathophysiology of spinal cord injury, we wonder if LPA could be involved in the pathophysiology of ALS. To assess whether activation of LPA2 contributes to ALS, we crossed LPA2 null with SOD1G93A mice. Our results reveal that the absence of LPA2 delays onset and progression of the disease and prevent the muscle atrophy in ALS mice. Collectively the results presented here provide more novel data that could underline new therapeutic strategies for ALS.

Amyotrophic lateral sclerosis (ALS) is a disease in which the motor neurons die in a progressive manner, leading to paralysis and muscle wasting. ALS is always fatal, usually through respiratory failure when the disease reaches muscles needed for breathing. Most cases are sporadic, but approximately 5–10% are familial. The first gene to be linked to familial ALS encodes the antioxidant enzyme superoxide dismutase-1 (SOD1). Today, more than 160 different mutations in SOD1 have been found in ALS patients.  The mutant SOD1 proteins cause ALS by gain of a toxic property that should be common to all. Aggregates of SOD1 in motor neurons are hallmarks of ALS patients and transgenic models carrying mutant SOD1s, suggesting that misfolding, oligomerization, and aggregation of the protein may be involved in the pathogenesis. SOD1 is normally a very stable enzyme, but the structure has several components that make SOD1 sensitive to misfolding. The aim of the work in this thesis was to study misfolded SOD1 in vivo. Small amounts of soluble misfolded SOD1 were identified as a common denominator in transgenic ALS models expressing widely different forms of mutant SOD1, as well as wild-type SOD1. The highest levels of misfolded SOD1 were found in the vulnerable spinal cord. The amounts of misfolded SOD1 were similar in all the different models and showed a broad correlation with the lifespan of the different mouse strains. The misfolded SOD1 lacked the C57-C146 intrasubunit disulfide bond and the stabilizing zinc and copper ions, and was prinsipally monomeric. Forms with higher apparent molecular weights were also found, some of which might be oligomers. Misfolding-prone monomeric SOD1 appeared to be the principal source of misfolded SOD1 in the CNS. Misfolded SOD1 in the spinal cord was found to interact mainly with chaperones, with Hsc70 being the most important. Only a minor proportion of the Hsc70 was sequestered by SOD1, however, suggesting that chaperone depletion is not involved in ALS.  SOD1 is normally found in the cytoplasm but can be secreted. Extracellular mutant SOD1 has been found to be toxic to motor neurons and glial cells. Misfolded SOD1 in the extracellular space could be involved in the spread of the disease between different areas of the CNS and activate glial cells known to be important in ALS. The best way to study the interstitium of the CNS is through the cerebrospinal fluid (CSF), 30% of which is derived from the interstitial fluid. Antibodies specific for misfolded SOD1 were used to probe CSF from ALS patients and controls for misfolded SOD1. We did find misfolded SOD1 in CSF, but at very low levels, and there was no difference between ALS patients and controls. This argues against there being a direct toxic effect of extracellular SOD1 in ALS pathogenesis. In conclusion, soluble misfolded SOD1 is a common denominator for transgenic ALS model mice expressing widely different mutant SOD1 proteins. The misfolded SOD1 is mainly monomeric, but also bound to chaperones, and possibly exists in oligomeric forms also. Misfolded SOD1 in the interstitium might promote spread of aggregation and activate glial cells, but it is too scarce to directly cause cytotoxicity.

Diseases affecting motor neurons, such as amyotrophic lateral sclerosis (Lou Gerhig's disease), hereditary spastic paraplegia and spinal bulbar muscular atrophy (Kennedy's disease) form a heterogeneous group of chronic progressive diseases and are among the most puzzling yet untreatable illnesses. Over the last decade identification of mutations in genes predisposing to these disorders has provided the means to better understand their pathogenesis. The discovery 13 years ago of SOD1 mutations linked to ALS, which account for less than 2% of all cases, had a major impact in the field. However, despite intensive research effort, the pathways leading to the specific motor neurons degeneration in the presence of SOD1 mutations have not been fully identified. The research projects presented here aim to investigate the role of different cell types and tissues in the pathology of SOD1-linked ALS, and to identify new genetic factors involved in sporadic ALS cases. LoxP transgenic mice expressing mutated G85R SOD1, allowing transgene expression in cell and tissue specific manner, have been successfully generated. However, mice, up to 2.5 years of age, did not develop any motor neuron deficits or any developmental abnormalities. We concluded that this might be due to insufficient level of the transgene expression in our transgenic animals. Also, a number of candidate genes for ALS have been identified, such as ALS2, VEGF, PRPH, CHGA and CHGB, based on their pattern of expression and biological function. These genes have been screened for mutations in a cohort of ALS patients and, we have identified one basepair deletion in the ALS2 and in the PRPH genes, and we have also found a strong genetic association between the CHGA and CHGB genes with ALS. An in-vitro cell transfection approach has been used to investigate the biological effects of mutations within the PRPH genes and, of particular interest, this technique has revealed the first functional variants in a neurofilament associated gene ever describe in ALS.

This PhD thesis examines the relationship between Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). ALS is a rapidly progressive neurodegenerative movement disorder characterized by muscle weakness, spasticity and abnormal reflexes. In a very small subset of patients (5-15%), ALS is associated with FTD. Furthermore, a larger subset of patients who do not suffer from overt dementia, develop subtle deficits in cognition and behaviour (up to 50%). The changes have mostly been observed in the domains of executive functions, language and behavioural functioning. These observations have led some researchers to propose a continuum of dysfunction between ALS and FTD, ranging from an absence of neuropsychological abnormalities to mild, subclinical changes to a profile consistent with a full-blown FTD-syndrome in ALS. FTD consists of three subsyndromes; the first ‘executive-behavioural’ type, frontal variant FTD (fvFTD), is predominantly characterized by behaviour abnormalities, difficulties with using strategies and social judgement. In contrast, the other two types mainly involve problems with ‘language’, including a central degradation of knowledge for words, objects, people (semantic dementia; SD) as well as complications with speaking, spelling and the sounds of language (progressive non-fluent aphasia; PNFA). The current study aims to explore whether the cognitive-behavioural deficits found in nondemented ALS-patients can be classified as subclinical forms of the first two FTDsyndromes, i.e. fvFTD and SD. In addition, the study further examined whether executive and language impairments co-exist or rather occur independently. To answer the research questions, a battery of neuropsychological tests was employed, adapted to patients’ speech and motor disabilities, as well as behavioural questionnaires. The data revealed there was evidence of both executive and language involvement characteristic of FTD, albeit to a subtle extent. ALS-patients showed deficits on a test of Theory of Mind (ToM). On this test, participants were asked to judge the thoughts and feelings of another, using the direction of eye gaze, a cue considered to be important for social interaction. Results indicated that ALS patients had difficulties with affective ToM, i.e. recognizing feelings of others, and this effect was not driven by perceptual or attentional difficulties. In addition, patients exhibited a subtle deficit with empathy as well as a range of behavioural abnormalities. Furthermore, ALS-patients showed abnormal performance on a complex multi-modal semantic association task which involved assigning the correct picture iii to the sound of an object. This central deficit emerged in the presence of normal audio-visual information processing and episodic memory functions. Moreover, a category-specific deficit for man-made objects was detected in patients. Individual case-analyses showed that various subsets of patients were impaired on the language and executive tasks. These analyses also showed that executive and language problems can occur independently as well as simultaneously in patients with ALS. In addition, analysis of individual cases revealed that some patients’ performance on the decision making tasks was similar to that found in patients with either orbitofrontal or dorsolateral dysfunction, while there was little if any evidence of a pattern of impairment similar to that seen with anterior cingulate dysfunction. The observed difficulties with social cognition and semantic processing indicate that executive and language problems, characteristic of the two FTD syndromes, can be detected in patients with classical ALS.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease. TDP-43 is found in pathological protein aggregates in neurons and glia in ALS and it is part of some mRNA transport granules. MBP messenger RNA (mRNA) must be transported to the oligodendrocyte processes for correct myelination. If TDP-43 were part of MBP mRNA transport granules, its aggregation could lead to loss of MBP in the CNS. Additionally, C9orf72 is a gene whose GGGGCC expansion mutation causes ALS. The expansion binds hnRNP-A2, a protein essential for the transport of MBP mRNA. This interaction may lead to the sequestration of hnRNP-A2, reducing its availability and causing a shortage of MBP in the CNS. Aims: To characterise the oligodendroglial pathology and the loss of MBP in our cohort of ALS cases, and to create zebrafish lines to investigate the development of oligodendroglial degeneration in sporadic ALS and C9orf72 ALS. The overarching hypothesis of the project is that the observed oligodendrocyte degeneration in ALS is primarily caused by a dysfunction of MBP mRNA transport, causing demyelination. Methods & Results: Using immunohistochemistry in human post mortem tissue, this project reports a greater, distinct ubiquitin-related glial pathology in the primary motor cortex of C9orf72-ALS cases. This C9orf72-related glial pathology was independent of the dipeptide-repeat protein inclusions usually found in the motor neurones of these patients. Using Western blot and qPCR, the levels of MBP and PLP, another myelin protein that is translated in the oligodendrocyte cell body, were measured. A reduction in the levels of MBP, but not those of PLP, was found in the spinal cord lateral corticospinal tracts of ALS cases, which was more pronounced in C9orf72 ALS. This reduction of the MBP levels was not accompanied by either a reduction in the number of axons panning that area or the levels of MBP mRNA. Conclusions: These results suggest that an impairment of mRNA transport is the main cause of the reduced MBP levels in the spinal cord of ALS patients and that oligodendrocyte degeneration precedes axonal degeneration in ALS. Were this confirmed using in vivo models of ALS, it would imply that ALS begins as a myelinopathy which later causes the apparition of neuronal pathology and death. None of the in vivo models trialled in this project were successful, but others have created promising models that will surely help understand oligodendroglial degeneration in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal neurodegenerative disease characterised by motor neuron degeneration and muscle denervation, atrophy and eventual paralysis. Approximately 10% of ALS cases are familial, caused by mutations in a range of genes including superoxide dismutase 1 (SOD1), TDP-43 (TARDBP) and fused-in-sarcoma (FUS), although the most common genetic cause of ALS is now thought to be a hexanucleotide repeat in C9ORF72. Mouse models of ALS, most commonly created by overexpressing wildtype or disease-causing mutant human proteins, have been critical for our understanding of ALS pathogenesis. In this Thesis I investigated a number of new mouse models that express ALS-causing mutations including mutant mice created through N-ethyl-N-nitrosourea (ENU) mutagenesis, which induces random point mutations in the mouse genome, resulting in expression of the mutant proteins at physiological levels, as well as a new transgenic mouse which over-expresses wildtype human FUS. In the first experiments, a mutant mouse carrying a D83G mutation in mouse Sod1, analogous to a pathogenic human mutation, was examined. Sod1D83G/D83G mice show motor neuron degeneration and axonopathy. Examination of cultured primary embryonic motor neurons revealed that Sod1D83G/D83G motor neurons have a reduced viability, a reduction in neurite outgrowth and significant deficits in mitochondrial function. In the second set of experiments, four different mice with ENU-induced mutations in TDP-43 were examined. These mice did not show any signs of neuromuscular dysfunction in vivo, or signs of pathology in vitro, assessed by neurite outgrowth and stress granule formation. Furthermore, crossing TDP-43 mutant mice with SOD1G93A transgenic mice did not modify disease progression. In the final set of experiments, transgenic mice overexpressing wildtype human FUS were investigated. In vivo assessment of neuromuscular function in homozygote mice at 10 weeks of age revealed significant pathological changes in muscle function and motor unit survival. These results characterise a novel ENU-induced mouse model of mutant SOD1-induced ALS and a new transgenic mouse model in which overexpression of wildtype human FUS causes a severe motor phenotype. However, ENU-induced mutations in mouse TDP-43 do not result in any overt motor phenotype.

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
MicroRNAs (miRNAs) are emerging as a primary mediator of gene regulation in many different cell types. There is increasing evidence that specific subsets of miRNA play a prominent role in the nervous system, both in development and in specific neurodegenerative diseases. This study aims to elucidate the role of microRNA in selective motor neuron death that is the hallmark of amyotrophic Lateral sclerosis (ALS). Pre-symptomatic time-point was chosen since the levels of miRNAs are highly likely to be altered as a secondary consequence of cell injury and death in ALS. Laser capture microdissection (LCM) was used to study miRNA profiles in motor neurons of spinal cord tissue from SOD1G93A mice, the best characterized model of ALS. In preliminary work, using miRNA specific chips we have identified 2 miRNAs which are dramatically upregulated before disease onset. In this study, high RNA quality was achieved from laser captured cells, which consist in a major advance towards obtaining meaningful results of these miRNAs expression in downstream applications. Despite LCM technology has become increasingly sophisticated; rapidly obtaining enough amount of starting material for downstream applications is still extremely challenging. The combination of this optimized technique with microarrays, followed by RT-qPCR may provide insights into potential contribution of microRNAs to progression of neurodegeneration of motor neurons in ALS.

Our knowledge of the genetic contribution to Amyotrophic Lateral Sclerosis (ALS) is rapidly growing, and there is increasing research into how ALS spreads through the motor system and beyond. This thesis examines how genetic and non-genetic factors in ALS influence its spread. The genetic methods employed were PCR, genotyping, AFLP, DNA sequencing and gene expression. The methods to examine spread were H&E staining, clinical history, age of onset (AOO), survival and health utility. Statistical procedures applied included regression analyses of genetic and non-genetic factors, maximum likelihood estimation of genetic-phenotype variance and health utility, RNA-sequence analyses, and differential gene expression analysis. I found (A) that variation in the ATXN2 gene contributes to ALS, as does variation in C9ORF72 after correcting for the known C9ORF72 pathological hexanucleotide repeat (HREM). (B) On comparing regions of the spinal cord, patterns of differential gene expression between ALS cases and controls appeared consistent with spread and pathology. Functional annotation clustering revealed these genes were mostly involved in blood vessel and angiogenin-like function, glycoprotein-based activity, and leukocyte activity. (C) A significant proportion of survival variance in ALS could be explained by genetic variance. There were SNPs that predicted survival and AOO, one showing epistasis with the C9ORF72 HREM. (D) When modelling ALS progression using staging and a clinical trial dataset, the time and duration of each stage was statistically predictable. Staging also predicted health utility and other functional and psycho- metrics. The spread and pathology in ALS spinal cord regions affected gene expression profiles, which is likely a consequence of genetic susceptibility in that region. Indicators of spread, AOO and survival, could be predicted using genotypes. Disease progression was predictable as measured by clinical staging and health metrics. In summary, ALS spread seems to occur at a fixed rate in an individual and is influenced by genetics.

Motor neurone disease/amyotrophic lateral sclerosis (MND/ALS) is established as a multisystem neurodegenerative disorder which includes a broad spectrum of cognitive and behavioural symptoms similar to those with behavioural variant frontotemporal dementia (FTD). Behavioural symptoms include perseveration, disinhibition and most commonly apathy. This thesis examined the impact of apathy in ALS, and specifically, its relationship to clinical, patient and caregiver outcomes. Initial studies designed to elucidate the clinical significance of apathy demonstrated that nearly half of patients presenting at a specialised ALS clinic exhibited apathy of varying levels of severity. Patients with moderate to severe apathy had a considerably shorter survival time than those with no apathy and mild apathy, even after taking into account clinical confounders. Comprehensive multidimensional assessment of apathy revealed that patients with apathy had lower quality of life (QOL), most pronounced in the achievements in life and community-connectedness domains compared to non-apathetic patients. Of the cognitive, behavioural and emotional symptoms of apathy, only the emotional symptoms were predictive of achievements in life and community-connectedness. For caregivers, higher levels of cognitive, behavioural and emotional symptoms of apathy were reported by high-burden compared to low-burden caregivers. Only the behavioural symptoms of apathy were associated with greater caregiver burden. In conclusion, the importance of assessing apathy and its severity as part of routine clinical practice is of prognostic and therapeutic importance. In particular, moderate to severe apathy at presentation may be indicative of more aggressive disease which warrants prompt intervention and support for patients and their caregivers. Psychological interventions designed to specifically target the emotional symptoms of apathy may be of particular value for improving patients’ QOL. For caregivers, strategies to manage behavioural deficits associated with apathy, including their own reactions towards these behaviours may mitigate caregiver burden.

Project1: Unraveling the impact of microRNA on Amyotrophic Lateral Sclerosis pathogenesis. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics. Recent studies have shown that microRNA have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence, in this study we test the hypothesis that misregulation of miRNA expression play a role in the pathogenesis of ALS. Hence, we exploited human neuroblastoma cell lines expressing SOD(G93A) mutation as tools to investigate the role of miRNAs in familiar ALS. To this end, we initially checked the key molecules involved in miRNAs biogenesis and processing on these cells and we found a different protein expression pattern. Subsequently, we performed a genome-wide scale miRNA expression, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a small group of up and down regulated miRNA, which are predicted to play a role in the motorneurons physiology and pathology. We measured this group of misregulated miRNA by qPCR on cDNA derived from (G93A) mice at different stage of disease and furthermore on cDNA derived from lymphocytes from a group of sporadic ALS patients. We found that mir-129-5p was up-regulated in cells, mice and in patients and we validated that HuD as mir129-5p target. It has been reported that ELAVL4/HuD plays a role in neuronal plasticity, in recovery from axonal injury and multiple neurological diseases. Furthermore, we generated stable cell line overespressing mir129-5p and we found a reduction in neurite outgrowth and in the expression of differentiation markers in compare to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating ELAVL4/HuD level. Project 2: FUS/TLS depletion leads an impairment of cell proliferation and DNA Damage Response. FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function hypothesis (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line we performed growth proliferation and survival assays. From these experiments emerged that FUS-depleted cells display alterations in cell proliferation. In order to explain this observation, we tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than control cells. Based on the notion that FUS interacts with the miRNA processing proteins (Morlando et al. 2012), to explain this phenotype, we looked at miRNAs expression and we found an up-regulation of mir-7. Interestingly, this up-regulation is also observed in cells that express the ALS-linked FUS R521C mutation. Finally, since an increasing number of work correlated FUS with DNA damage and repair we explored the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). We found that FUS depletion had an effect on the initial level of DNA damage by inducing the phosphorylation of H2AX in basal condition and that it delayed DSB repair when acute DNA damage occurs. Interestingly, genotoxic treatment resulted in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress. Taken together, these studies may contribute to the knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal neuromuscular disease characterized by selective motorneurons loss. Since mutations in TARDBP and FUS genes were discovered to cause familial form of ALS and TDP-43 and FUS proteins play important roles in RNA metabolism, transcriptional alterations emerged as potential pathogenic mechanism. RNA metabolism include several aspects of RNA regulation such as RNA transcription, maturations and regulation. In this study we have investigated two different fields of RNA metabolism: the first one concerns to microRNAs (miRNA) which regulate translation of several mRNAs, and the second one is related to a specific muscular and neuronal transcription factor potential involved in ALS. First, we have assessed any selected miRNAs with neuronal functions in human neuroblastoma cell lines expressing the pathological SOD1(G93A) mutation and we found a small group of altered miRNAs. Subsequently, we explored these miRNAs in the spinal cord of transgenic SOD1(G93A) mice identified a panel of targets commonly altered in SOD1 ALS models. Furthermore, we assessed the expression levels of a panel of selected miRNAs in circulating cells obtain from patients affected by sporadic ALS form (sALS). This approach let us to identify two microRNAs (miR129-5p and miR200c) that were up-regulated in both SOD1 ALS models and in blood cells of patients with sporadic form of disease, evidencing two possible parameters potentially involved in the pathogenesis of both the sporadic and the familial form of ALS. Moreover, we also identified HuD protein as a potential molecular target of miR129-5p; this protein has been previously reported to play a role in neuronal plasticity and in recovery from axonal injury. Indeed, in a cell line stably overespressing mir129-5p we found a reduction in neurite outgrowth and decreased expression levels of differentiation markers with respect to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating HuD levels. In the second part of the study we investigated the possible involvement of two members of myocyte enhancer factor 2 (MEF2) family in the pathogenesis of ALS. MEF2D and MEF2C are transcriptional factors playing crucial roles both in muscle and in neuron development and maintenance. We have performed gene expression analysis in peripheral blood mononuclear cells (PBMCs), we showed a strong increased in MEF2D and MEF2C levels both in sporadic and in familial ALS (SOD1+) patients and a direct correlation between MEF2D and MEF2C mRNA levels was observed in patients and controls. Although protein levels were unchanged, a different pattern of distribution for MEF2D-MEF2C proteins in patient cells was found, suggesting a possible lack of their function. To evaluate the transcriptional activity of MEF2 proteins mRNA levels of their downstream targets BDNF, KLF6, RUFY3 and NPEPPS were assessed. Our results showed a significant down-regulation of BDNF, KLF6 and RUFY3 levels confirming that transcriptional activity of both MEF2D and MEF2C isoforms was altered in sporadic and familial ALS patients. In conclusion, our results evidenced a systemic alteration of MEF2D and MEF2C pathways in ALS patients independently from the presence of SOD1 gene mutations, highlighting a possible common feature between the sporadic and the familiar form of disease which are characterized by a different clinical phenotype and pathological hallmarks.

L’esclerosi lateral amiotròfica (ELA) és una malaltia neurodegenerativa que causa la paràlisi i la mort dels pacients arrel de la pèrdua de les motoneurones de la medul·la espinal i el cervell. Malauradament, els únics tractaments actuals que hi ha són pal·liatius i no endarrereixen la progressió de la malaltia. Una de les característiques de l’ELA és l’activació aberrant del sistema immunològic: (i) activació de les cèl·lules glials (microglia i astròcits) a nivell del sistema nerviós central i (ii) infiltració dels leucòcits, principalment macròfags, al sistema nerviós perifèric. La resposta inflamatòria és un procés fisiològic que s’encarrega de l’eliminació de detritus cel·lulars així com de l’activació de processos de reparació tissular en teixits que han patit un dany o infecció. No obstant, les cèl·lules immunològiques també poden secretar mediadors citotòxics que causen danys i fins i tot la mort de les cèl·lules circumdants. La regulació d’aquests processos però, es deu a la presencia de diverses molècules que es troben en el teixit afectat. En l’ELA, diversos estudis han demostrat que aquesta resposta inflamatòria no té un rol protector, sinó deleteri. Basant-nos en aquestes evidències, en la present tesi doctoral vam avaluar si la modulació de la resposta inflamatòria podia millorar la progressió de la malaltia. En concret, ens vam centrar en l’estudi de tres dianes inflamatòries: (i) el receptor del factor estimulador de colònies tipus 1; (ii) la citocina anti-inflamatòria, interleuquina-37; (iii) el lípid immunoresolutor, Maresina-1. En aquest treball, mostrem que la modulació de la resposta inflamatòria per les tres vies confereix neuroprotecció i endarrereix el curs clínic de l’ELA.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive paralysis and death to patients due to the degeneration of motor neurons in the spinal cord and the brain. At present, therapy is mainly symptomatic and fails to halt disease progression. A common feature of ALS, and other neurological disorders, is the occurrence of an inflammatory reaction consisting of activated glial cells (microglia and astrocytes) within the central nervous system, and leukocytes, mainly macrophages, in the peripheral nerves. The inflammatory response is a physiological process with very precise control and plays an essential role in the removal of cell debris and the activation of repair processes in infected or injured tissues. However, immune cells also secrete cytotoxic mediators that exert damage in healthy neighboring cells and even lead to cell death. This dual sword edge of immune cells likely depends on regulatory mediators that are present in the milieu. However, in ALS, as well as, in other neurological conditions inflammatory response is believed to trigger greater hazardous than protective actions. Based on these evidences, in the present thesis we aimed at assessing whether modulation of key aspects of inflammation could ameliorate the clinical course of ALS disease. In particular, we have focused our interest in three main targets: (i) the proinflammatory colony stimulating factor 1 receptor; (ii) the anti-inflammatory cytokine, interleukin-37; (iii) the immunoresolvent agent, Maresin-1. We provide novel data demonstrating that these approaches confer neuroprotection against the clinical course of ALS disease.

Amyotrophic Lateral Sclerosis (ALS) is a lethal motor neuron disorder, characterized by selective and progressive degeneration of both upper and lower motor neurons. Currently the only available drug for the treatment of ALS is Riluzole, which exerts little overall effects. Therefore ALS is currently an untreatable disease. A small proportion (5%) of patients develop the hereditary form of the disease known as familial ALS (FALS), 40% of which are associated with G4C2 hexanucleotide repeat expansion in the C9orf72 gene and 20% with mutations in the SOD1 (copper/zinc superoxide dismutase-1) gene. In addition, recently a mutation has been identified at position R199W in the DAO gene, accounting for less than 1% of FALS cases. The DAO gene encodes D-amino acid oxidase (DAO) protein, a peroxisomal flavin adenine dinucleotide (FAD)-dependent oxidase involved in degradation of D-amino acids. There have been reports indicating that DAO protein plays a crucial role in regulation of D-serine (a D-amino acid involved in neuronal signalling) in the central nervous system. The focus of this thesis was to investigate and establish the role of both mutant (DAOR199W) and wild type DAO proteins (DAOWT) in ALS pathology in terms of disease onset, disease progression and survival in a well-known model of ALS, the high copy number SOD1G93A mouse, through two large cohort survival studies. Thus, for the first survival study (cross of DAOR199W and SOD1G93A) we hypothesized that overexpression of the mutant allele exacerbates the ALS-like phenotypes, resulting in an earlier onset, accelerated progression and shorter lifespan in the double transgenic animals. Our data from this study showed that DAOR199W expression does not modify disease onset, progression and survival in the DAOR199W/SOD1G93A animals compared to their SOD1G93A littermates. The second survival study (cross of DAOWT and SOD1G93A) was designed based on reports of elevated levels of the D-serine in the spinal cord of ALS patients. Considering the high abundance of D-serine in the central nervous system and its ability to induce excitotoxicity, we hypothesized overexpression of the DAOWT enzyme results in a more rapid break down of D-serine, ameliorating the SOD1G93A phenotype by delaying disease onset, slowing down disease progression and prolonging survival in the double transgenics. Our results from this survival study indicate that overexpression of DAOWT at the cellular level does not effect disease onset but significantly delays the onset of neurological symptoms, slows down progression and prolongs survival in both sexes in the SOD1G93A/DAOWT animals, compared to their SOD1G93A littermates. In a separate approach we aimed to investigate the affect of two different murine IGF- I isoforms (IGF-IA and MGF), in in vivo models of ALS by means of an efficient method of gene delivery, electroporation of recombinant plasmid vectors. For this investigation we hypothesized treating SOD1G93A animals with IGF-I, results in slower disease progression and prolonged survival. Both vectors were successfully cloned, expressed in cell culture followed by in vivo electroporation. However there was insufficient time to test their therapeutic potential in the SOD1G93A mouse.

In this study a candidate-generating approach was first employed, using proteomics to identify mitochondrial proteins whose expression changed in the presence of mutant SODI in a wellvalidated model of SOD I-related familial amyotrophic lateral sclerosis (ALS). Proteins whose expression changed in a mutant-specific fashion had functions potentially relevant to the pathogenesis of ALS including roles in apoptosis, protein processing and anti-oxidant defence. I then validated selected protein changes of interest by Western blotting in mitochondrial preparations of further NSC34 cells and G93A transgenic mouse spinal cord. Peroxiredoxin 3 (Prx 3), a thioredoxin-dependent hydroperoxidase, was down-regulated in NSC34 cells expressing two different species of mutant SOD I and in 90 d G93A transgenic mouse whole spinal cord. Immunostaining for Prx 3 and the known mitochondrial matrix protein HSP60 in NSC34 cells revealed co-localizing staining patterns, confirming the expected mitochondrial localization of Prx 3. Prx 3 immunohistochemistry confirmed its expression within the mitochondria of human and murine spinal motor neurons, establishing potential relevance to the human disease. Q-PCR was then used to show a down regulation of Prx 3 mRNA in spinal motor neurons from patients with both sporadic and SOD I-related FALS. The small drug molecule ebselen was identified as a peroxiredoxin mimic and was shown selectively to protect motor neuronal cells expressing two different species of mutant human SOD I against apoptosis induced by withdrawal of serum. Finally, having postulated that ebselen may act by up-regulating anti-oxidant protein transcription as well as acting as an anti-oxidant in its own right, ebselen was shown to induce transcription of the anti-oxidant response element (ARE), an oxidative stress-sensitive promoter sequence common to many anti-oxidant response genes, including members of the peroxiredoxin family and the proteins which maintain them in their active, reduced state. Examination of the sequence 5' to the Prx 3 gene revealed a putative ARE response element.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to respiratory compromise and eventually death within two to five years. Even though people with ALS must make many treatment decisions, none has such a significant impact on quality of life and survival as the one pertaining to assisted ventilation. A qualitative research study was undertaken to elicit factors that are pertinent to this decision-making process. Ten individual, semi-structured interviews were conducted with individuals with ALS. Six main themes emerged from the interviews. These are: meaning of the intervention, the importance of context, values, and fears in decision-making, the need for information, and adaptation/acceptance of the intervention. Based on these findings, it is argued that a pluralistic conception of autonomy as well as a shared decision-making model is better suited to give high priority to patient autonomy in this context. Some recommendations to improve clinical practice are proposed.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with considerable clinical, genetic and pathological overlap. A GGGGCC hexanucleotide repeat expansion in a non-coding region of C9orf72 on chromosome 9 is the major cause of both FTLD and ALS. An understanding of the mechanisms through which the expansion leads to neurodegeneration will therefore be vital for development of novel therapeutics. There are 3 possible mechanisms through which the GGGGCC expansion may cause toxicity: (i) through haploinsufficiency of C9orf72, (ii) repetitive RNA transcripts arising from the expansion may be toxic or (iii) translation of the expansion may produce toxic peptides. Whilst the expansion is located in a non-coding region of the gene, long GC-rich RNA transcripts may be translated in the absence of an ATG start codon, through a process known as repeat-associated non-ATG translation (RAN-translation). 5 distinct dipeptide repeat proteins (DPRs) have been found to arise from the expansion through RAN-translation of the sense and antisense strands in all frames: poly-GA, -GR, -PR, -AP and –GP. All 5 DPRs have been shown to aggregate in patient tissue, indicating that they may play a role in C9FTLD/ALS pathogenesis. This project aimed to generate a series of models to investigate the mechanisms of neurodegeneration in C9FTLD/ALS, using C. elegans and cell culture. A transgenic worm strain which does not express the C. elegans orthologue of C9orf72 was first characterised. No impairments were observed in motility or life-span, demonstrating that loss of C9orf72 function does not cause an ALS-like phenotype in C. elegans. When considered alongside recent literature, this finding suggests that toxic gain of function mechanisms may be more important in C9FTLD/ALS pathogenesis. The impact of DPRs on cellular function was next investigated. Constructs were generated containing alternative codon sequences for each of the 5 DPRs, to express each peptide in the absence of the repetitive GGGGCC RNA sequences found in disease. A step-wise cloning strategy was employed to progressively increase repeat-length in these constructs until physiologically-relevant lengths of > 1000 repeats were obtained. DPRs were then expressed in HeLa cells, in order to assess the individual effects of each peptide on cellular function. Poly-GA formed large, star-shaped cytoplasmic inclusions which co-localised with ubiquilin-2 and p62, closely resembling the inclusions observed in patient tissue. This implies a potential role of proteasome dysfunction in C9FTLD/ALS. Conversely, the alanine-rich DPRs, poly-GR and –PR, translocated to the nucleolus, where poly-GR in particular caused nucleolar stress. Furthermore, nucleolar poly-GR caused loss of Cajal bodies from the nucleus, and a loss or mislocalisation of survival motor neuron protein (SMN). This is of particular interest to C9ALS, since loss of SMN is selectively toxic to motor neurons. Furthermore, loss of Cajal bodies and nucleolar stress are likely to cause defects in RNA processing, which may contribute to neurotoxicity.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurological disorder caused by the selective degeneration of upper and lower motor neurons, for which there are currently no effective treatments. 10% of ALS cases are familial, of which 15-20% are caused by mutations in the copper/zinc superoxide dismutase gene (SOD1). The primary triggers for motor neuron degeneration in ALS are unknown, but research in patients and SOD1 models has revealed several mechanisms which may contribute. These include: oxidative stress, mitochondrial abnormalities, inflammation and protein aggregation. This study focused on the pre-clinical testing of two-potential therapeutics for ALS in the SOD1G93A murine model of the disease; metformin, an anti type II diabetes drug which has been shown to have anti-inflammatory and anti-oxidant properties and the ability to bring about mitochondrial biogenesis, and trehalose, a chemical chaperone and enhancer of autophagy, which has been shown to reduce protein misfolding and aggregation. We performed an initial study in which oral metformin administration from 35 days increased the survival of functional motor units in the hindlimbs of male and female SOD1G93A mice at 100 days. Consequently we performed a dose-response survival study in SOD1G93A mice with longitudinal monitoring of weight and neurological score. Surprisingly, metformin had no effect in males and brought about a dose-dependent negative effect on the onset of neurological symptoms and on disease progression in females. We hypothesise this negative effect may have resulted from a metformin-induced reduction in oestrogen production. Oral trehalose administration was tested using the same survival study format. Although trehalose treatment brought about a dose-dependent delay in weight-loss in males, it had no effect on the onset or progression of neurological symptoms or on survival in male or female mice. We conclude that neither metformin nor trehalose represent strong candidates for clinical trial in ALS patients when administered orally.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that leads to decreased muscle function resulting in problems with movement, breathing, swallowing, and speech. In the United States, approximately 5,600 people are diagnosed with ALS annually (ALS Association [ALSA], 2010). ALS can attack muscles all over the body, including those in the velopharynx, a muscular valve-like structure located at the back of the oral cavity. In a healthy adult, the velopharynx closes off the passageway to the nasal cavity during speech production to direct air through the mouth rather than through the nose (except during productions of /m/, /n/, and /ng/). Speech that is produced with an open velopharynx sounds indistinct and has a nasal quality. Many people with ALS experience difficulty producing clear, articulate speech because their velopharynx remains open for the majority of speech sounds. As a result, their speech eventually becomes unintelligible. Currently, there is little information available about the function of the velopharynx in people with ALS. The purpose of this study is to describe velopharyngeal function during speech in people with ALS. This information will be compared with their speech rate and intelligibility scores, both common measures used to document speech decline.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive paralysis of limbs and bulbar musculature. This severe physical impairment makes cognitive evaluation a big challenge, thus there is a great need for an assessment that does not require overt motor responses. Moreover, we need of augmentative communication strategies because the disease generally leads to complete paralysis and, therefore, patients are unable to communicate with the external world by any means. For this purpose, Brain Computer Interfaces (BCIs) seem a promising approach to facilitate communication with these patients. The aim of this thesis is twofold. First, assessing cognitive processing in ALS by means of a novel evaluation tool. Second, allowing brain communication in completely paralyzed ALS patients who had lost their vision in order to eliminate the unbearable loss of communication in paralysis (“unlocking the locked-in”). The first study introduces a novel approach for assessing cognitive functions in ALS. This approach uses neuropsychological tests that require minimal overt motor or verbal responses; together with vibro-tactile P300s. Results indicate mild cognitive impairment in oral language comprehension tasks and reduced vibro-tactile P300 amplitudes in patients compared to healthy controls. Importantly, correlations between the vibro-tactile P300 latency and psychometric test results suggest that the former measure could serve as a neurophysiological marker of cognitive decline in ALS patients. The second study introduces a distraction paradigm based in auditory event-related potentials (ERPs) to evaluate the ability of change detection, focusing, and re-orientation of attention in ALS. The results revealed a modification of the amplitude and the latency of the N200, the P300 and the re-orienting negativity (RON) components. This could suggest an alteration of the endogenous mechanism that controls the detection of change, thus resulting in a reduction of the allocation and the re-orientation of attentional resources. The third study aimed at testing the feasibility of a Near Infrared Spectroscopy (NIRS) -based BCI communication approach for patients in the Completely Locked-in Stage (CLIS) due to ALS. For this purpose two CLIS patients were trained to control their cerebral-cortex´s functional-activations in response to auditory processing of correct or incorrect statements assessed with NIRS. The results of the study are very promising, showing that both CLIS patients communicated with fronto-cortical oxygenation based BCI at an average correct response rate of 70% over a period of several weeks. We conclude that this novel approach of brain-communication is safe and, reliable, representing, so far, the best communication possible for patients in completely locked-in state. In conclusion we propose a) the novel combination of vibro-tactile or acoustic ERPs and motor-independent neuropsychological tests as an alternative and easily implementable way for assessing cognitive functions in ALS and b) we confirm the usefulness and effectiveness of above mentioned electrophysiological approaches in the late stage of ALS either to assess cognitive processing or to establish communication with a BCI system.
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive paralysis of limbs and bulbar musculature. This severe physical impairment makes cognitive evaluation a big challenge, thus there is a great need for an assessment that does not require overt motor responses. Moreover, we need of augmentative communication strategies because the disease generally leads to complete paralysis and, therefore, patients are unable to communicate with the external world by any means. For this purpose, Brain Computer Interfaces (BCIs) seem a promising approach to facilitate communication with these patients. The aim of this thesis is twofold. First, assessing cognitive processing in ALS by means of a novel evaluation tool. Second, allowing brain communication in completely paralyzed ALS patients who had lost their vision in order to eliminate the unbearable loss of communication in paralysis (“unlocking the locked-in”). The first study introduces a novel approach for assessing cognitive functions in ALS. This approach uses neuropsychological tests that require minimal overt motor or verbal responses; together with vibro-tactile P300s. Results indicate mild cognitive impairment in oral language comprehension tasks and reduced vibro-tactile P300 amplitudes in patients compared to healthy controls. Importantly, correlations between the vibro-tactile P300 latency and psychometric test results suggest that the former measure could serve as a neurophysiological marker of cognitive decline in ALS patients. The second study introduces a distraction paradigm based in auditory event-related potentials (ERPs) to evaluate the ability of change detection, focusing, and re-orientation of attention in ALS. The results revealed a modification of the amplitude and the latency of the N200, the P300 and the re-orienting negativity (RON) components. This could suggest an alteration of the endogenous mechanism that controls the detection of change, thus resulting in a reduction of the allocation and the re-orientation of attentional resources. The third study aimed at testing the feasibility of a Near Infrared Spectroscopy (NIRS) -based BCI communication approach for patients in the Completely Locked-in Stage (CLIS) due to ALS. For this purpose two CLIS patients were trained to control their cerebral-cortex´s functional-activations in response to auditory processing of correct or incorrect statements assessed with NIRS. The results of the study are very promising, showing that both CLIS patients communicated with fronto-cortical oxygenation based BCI at an average correct response rate of 70% over a period of several weeks. We conclude that this novel approach of brain-communication is safe and, reliable, representing, so far, the best communication possible for patients in completely locked-in state. In conclusion we propose a) the novel combination of vibro-tactile or acoustic ERPs and motor-independent neuropsychological tests as an alternative and easily implementable way for assessing cognitive functions in ALS and b) we confirm the usefulness and effectiveness of above mentioned electrophysiological approaches in the late stage of ALS either to assess cognitive processing or to establish communication with a BCI system.

L’esclerosi lateral amiotròfica (ELA) és una malaltia neurodegenerativa devastadora sense cap tractament efectiu disponible. Els mecanismes moleculars involucrats en la mort de les motoneurones (MN) són complexos i inclouen nombroses disfuncions d’aquestes, on les cèl·lules del voltant com els astròcits i la micròglia hi poden contribuir. La Neuregulina 1 (NRG1) és un factor neurotròfic expressat en les MNs i les unions neuromusculars que ajuda en el desenvolupament neuromuscular i axonal i en el seu manteniment. Estudis recens suggereixen un rol crucial de la NRG1 i dels seus receptors ErbB en l’ELA, particularment per la isoforma I (NRG1-I) en el procés de reinnervació colateral, i per la isoforma III (NRG1-III) en la preservació de les MNs, obrint un nou camp per desenvolupar noves teràpies per l’ELA. Tanmateix, per clarificar el rol de la via NRG1-ErbB en la supervivència de les MNs és necessari fer més estudis i així provar la seva eficàcia terapèutica. En la present tesis, hem avaluat l’efecte terapèutic de la sobreexpressió de la NRG1 en el sistema nerviós central i perifèric. D’aquesta manera, primer van caracteritzar el rol de la NRG1 exògena utilitzant un model in vitro de cultius organotípics de medul·la espinal subjectes a una excitotoxicitat crònica causada per l’àcid DL-threo-β-hydroxaspàrtic. Els nostres resultats mostren que l’addició en el medi de la NRG1 recombinant humana (rhNRG1) va incrementar significativament la supervivència de les MNs a través de l’activació dels receptors ErbB. Aquest efecte va ser bloquejat quan vam afegir el lapatinib, un inhibidor dels ErbB. El tractament amb la rhNRG1 també va reduir la reactivitat microglial superant els efectes causats per l’excitotoxicitat, i va activar la via de supervivència PI3K/AKT, a més de restaurar el flux autofàgic normal en el cultiu. A més a més l’addició de la rhNRG1 al medi va promoure el creixement neurític motor i sensorial. A continuació vàrem decidir d’utilitzar teràpies gèniques basades en vectors adeno-associats per sobreexpressar la NRG1-I en el múscul esquelètic, i la NRG1-III en la medul·la espinal per preservar les MNs en un model in vivo d’ELA, els ratolins SOD1G93A. Els resultats obtinguts van indicar que ambdues teràpies gèniques van ser capaces de preservar la funció neuromuscular dels músculs de les extremitats, van millorar la locomoció, van incrementar el nombre de MNs supervivents, i van reduir la reactivitat astrocitària i microglial en les femelles SOD1G93A tractades a l’estadi final de la malaltia. A més a més, en la medul·la espinal l’eix NRG1-III/ErbB4 regula l’excitabilitat de la MN a través del transportador KCC2 i redueix l’expressió del marcador de vulnerabilitat de MN MMP-9. La NRG1-I expressada en el múscul esquelètic va senyalitzar amb els receptors ErbB2 i 3 presents en les cèl·lules de Schwann terminals, per promoure reinnervació axonal. Malgrat això, quan vàrem combinar ambdues teràpies gèniques no vam trobar un efecte sinèrgic. Conjuntament, els nostres resultats indiquen que les isoformes de la NRG1 juguen un rol important en la supervivència de les MN i que la sobreexpressió viral pot ser considerada com una nova teràpia per tractar l’ELA.
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder with no effective treatment currently available. The molecular mechanisms that are involved in the motoneuron (MN) death are complex and include several MN dysfunctions, and contribution of surrounding cells such as microglia and astrocytes. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that supports axonal and neuromuscular development and maintenance. Recent studies have suggested a crucial role for NRG1 and their ErbB receptors in ALS, particularly for isoform I (NRG1-I) in the collateral reinnervation process, and isoform III (NRG1-III) in the preservation of the MNs, opening a new window for developing novel ALS therapies. However, further studies are needed to clarify the role of the NRG1-ErbB pathway on MN survival and to provide the proof of concept of its therapeutical efficacy. In the present thesis we have evaluated the therapeutic effect of NRG1 overexpression in the central and the peripheral nervous system. For this purpose, we first characterized the role of exogenous NRG1 using an in vitro model of spinal cord organotypic cultures (SCOC) subject to chronic excitotoxicity caused by DL-threo-β-hydroxyaspartic acid. Our results revealed that addition of recombinant human NRG1 (rhNRG1) to the medium significantly increased MN survival through the activation of ErbB receptors, which was blocked by addition of lapatinib, an ErbB inhibitor, and reduced microglial reactivity overcoming the excitotoxicity effects. rhNRG1 activated the pro-survival PI3K/AKT pathway and restored the autophagic flux in the spinal cord culture. Furthermore, addition of rhNRG1 to the medium promoted motor and sensory neurite outgrowth. We have then directed gene therapies based on adeno-associated viruses to overexpress NRG1-I in the skeletal muscles, and NRG1-III in the spinal cord to preserve the MNs in the in vivo model of ALS, the SOD1G93A mice. Our results indicate that both gene therapies were able to preserve the neuromuscular function of the hindlimb muscles, improve the locomotor performance, increase the number of surviving MNs and reduce the astrocyte and microglial reactivity in the treated female SOD1G93A mice at the end-stage of the disease. Furthermore, in the spinal cord the NRG1-III/ErbB4 axis regulates MN excitability through the KCC2 transporter and reduces the expression of the MN vulnerability marker MMP-9. NRG1-I expressed in the skeletal muscle signals with ErbB2 and 3 receptors present in terminal Schwann cells to promote axonal reinnervation. However, when we aimed to combine both viral-mediated therapies we did not find a synergic effect. Altogether, our results indicate that NRG1 isoforms play an important role on MN survival and that a viral-mediated overexpression may be considered as a potential novel therapy to treat ALS.

Thesis (MHSc--Health Science) -- Auckland University of Technology, 2003.
Appendices A and B are not included in e-thesis. Also held in print (148 leaves, 30 cm.) in Akoranga Theses Collection (T 616.83 BRO)

L’Esclerosi Lateral Amiotròfica (ELA) és una malaltia neurodegenerativa de la motoneurona. Totes les neurones del sistema motor es veuen afectades pel flux degeneratiu en aquesta malaltia des de l’escorça motora primària fins a la junta neuromuscular. Al 1993, la descoberta de mutacions en el gen SOD1 va obrir nous horitzons experimentals amb la creació dels primers rosegadors transgènics per aquesta malaltia. Des d’aquell moment i fins a l’actualitat la mutació més estudiada en l’ELA ha estat la SOD1-G93A a tot el món. Els models transgènics per aquesta mutació de la SOD1 han revelat mecanismes essencials de la neurodegeneració en aquesta malaltia incloent l’excitotoxicitat, la disfunció proteica i la degeneració axosinàptica entre altres. En aquest treball hem explorat els canvis moleculars que tenen lloc als terminals-C, uns terminals molt especialitzats en les α-moto neurones, dels rosegadors transgènics SOD1-G93A. A més, també hem focalitzat la nostra atenció a la relació patològica que s’estableix en l’ELA familiar (ELAF) entre la mutació SOD1-G93A i les mitocòndries de les motoneurones. En relació als terminals C en moto neurones durant la ELAF, hem trobat canvis associats a l’aparició dels símptomes com ara expressió incrementada del factor neurotròfic Neuregulina-1 localitzat també per primer cop a la cisterna subsinàptica dels terminals C aposats a les α-moto neurones. La Neuregulina-1 en aquestes estructures de reticle endoplasmàtic va ser observada a dins de vesícules extracel·lulars (VEs), suggerint que l’anàlisi de la Neuregulina-1 en VEs durant ELA és especialment prometedor com a biomarcador potencial en aquesta malaltia. Així nosaltres hem desenvolupat també un nou mètode per tal d’aïllar VEs, donat que aquest és un pas essencial previ a l’estudi de les proteïnes associades amb aquestes estructures. El nostre mètode aplicat a la purificació de VEs en teixits complexos fou capaç de facilitar la identificació de la Neuregulina-1 en VEs provinents de teixits clínics i fluids biològics. En relació a les implicacions de la mitocòndria en la ELA, hem trobat que la mutació SOD1-G93A estabilitza la proteïna PINK1 a la mitocòndria seguidament activant el factor nuclear NFκB en neurones. La interacció seqüencial entre la SOD1 mutant i NFκB crea una clara disfunció en la capacitat proteolítica del proteosoma, el qual promou coagregació de la SOD1 mutant i el PINK1 en aquestes cèl·lules. Aquests resultats afegeixen un substancial coneixement mecanístic sobre els rols de la mitocòndria en els events neurodegeneratius clàssics de l’ELA, com ara en l’agregació de proteïnes disfuncionals en moto neurones. Seguint el nostre estudi de l’afectació mitocondrial en la ELA, hem creat i caracteritzat un nou model de Drosophila que expressa la mutació humana SOD1-G93A exclusivament en fibres musculars toràciques sota el promotor 24B. Aquest model de Drosophila transgènica recapitula amb èxit el fenotip mitocondrial prèviament observat de l’ELA presentant importants avantatges sobretot en l’elecció de nous compostos terapèutics. En definitiva, els resultats generats en aquesta tesi proporcionen evidència experimental, extensa comprensió molecular i insinuen nous horitzons terapèutics sobre els mecanismes moleculars i els events neurodegeneratius associats a la disfunció sinàptica i mitocondrial en l’ELAF.
La Esclerosis Lateral Amiotrófica (ELA) es una enfermedad neurodegenerativa de la motoneurona. Todas las motoneuronas se ven afectadas desde la corteza motora primaria hasta la unión neuromuscular. En 1993 la descubierta de mutaciones en el gen SOD1 abrió nuevos límites experimentales con la creación de los primeros roedores transgénicos para esta enfermedad. Desde ese momento y hasta la actualidad, la mutación más estudiada en la ELA ha sido la mutación SOD1-G93A. Los modelos transgénicos de esta mutación han revelado mecanismos esenciales de la neurodegeneración en la ELA, incluyendo la excitotoxicidad, la disfunción proteica y la degeneración axosináptica entre otras. En este trabajo hemos explorado los cambios moleculares que tienen lugar en los terminales C, unos terminales altamente especializados de las α-motoneuronas, en un modelo murino de ELA con la mutación SOD1-G93A. Además, también hemos focalizado nuestra atención sobre la relación patológica que se establece en la ELA familiar (ELAF) entre la mutación SOD1-G93A y las mitocondrias. En relación a los terminales C durante la ELAF, hemos encontrado cambios asociados con la aparición de síntomas, como por ejemplo el incremento de la expresión del factor neurotrófico Neuregulina-1, localizado por primera vez en la cisterna subsináptica de los terminales C. La Neuregulina-1 en esas estructuras de retículo endoplasmático fue observada dentro de vesículas extracelulares (VEs), sugiriendo que el análisis de la Neuregulina-1 dentro de VEs en la ELA resulta especialmente prometedor como biomarcador potencial para esta enfermedad. Así, nosotros hemos desarrollado también un nuevo método para purificar VEs, dado que este es un paso esencial previo al estudio de las proteínas asociadas con estas estructuras. Nuestro método aplicado a la purificación de VEs de tejidos complejos fue capaz de facilitar la identificación de la Neuregulina en VEs provenientes de tejidos clínicos y fluidos biológicos. En relación a las implicaciones de la mitocondria en la ELA, hemos encontrado que la mutación SOD1-G93A estabiliza la proteína PINK1 en las mitocondrias activando el factor nuclear NFκB en neuronas. La interacción secuencial entre la SOD1 mutante y el NFκB crea una clara disfunción sobre la capacidad proteolítica del proteosoma, la cual a su vez promueve co-agregación de la SOD1 mutante y PINK1 en estas células. Estos resultados suman un sustancial conocimiento mecanístico sobre los roles de la mitocondria en eventos degenerativos clásicos de la ELA, como es la agregación de proteínas disfuncionales en motoneuronas. Siguiendo nuestro estudio de la afectación mitocondrial en la ELA, hemos creado y caracterizado un nuevo modelo de Drosophila que expresa la mutación humana SOD1-G93A en fibras musculares torácicas bajo el promotor 24B. Este modelo de Drosophila transgénica recapitula con éxito en fenotipo mitocondrial característico de la ELA presentando importantes ventajas para la elección de nuevos compuestos terapéuticos. En definitiva, los resultados generados en esta tesis proporcionan evidencia experimental, extensa comprensión molecular y insinúan nuevos horizontes terapéuticos acerca de los mecanismos moleculares y eventos neurodegenerativos asociados con la disfunción sináptica y la disfunción mitocondrial en la ELAF.
Amyotrophic Lateral Sclerosis (ALS) is an orphan age-associated neurodegenerative disease. All motoneurones in ALS are affected by degenerative flow from the primary motor cortex to the neuromuscular junction. In 1993, mutations of the gene SOD1 opened new research avenues allowing for the generation of familial ALS experimental models in rodents. Since then, the FALS mutation SOD1-G93A has been extensively studied worldwide in ALS to date. Transgenic models for this SOD1 mutation have revealed essential mechanisms of neurodegeneration including excitotoxicity, proteinopathy and axosynaptic degeneration among others. In this dissertation, we explored the molecular changes that occur in C-terminals, a very specialised synapse type from α-motoneurones of SOD1-G93A rodents. Also, we focused on the pathological relationship between the FALS mutant SOD1-G93A and mitochondria in motoneurones. With regard to C-terminals in FALS motoneurones, we found changes that were symptomatically associated with the up-regulated expression of the neurotrophic factor Neuregulin-1 located for the first time in the subsurface system of C-boutons juxtaposed to α-motoneurones. Furthermore, Neuregulin-1 in these endoplasmic reticulum structures was observed inside extracellular vesicles, suggesting that analysis of Neuregulin-1 from extracellular vesicles in ALS holds promise as a potential reliable biomarker for that neurodegenerative disease. We therefore have developed a new method for isolation of extracellular vesicles, as this remains as an essential step for the study of molecules associated with these structures. Our method applied to purify extracellular vesicles from complex biological tissues was able to facilitate the identification of Neuregulin-1 in extracellular vessicles from clinical tissues and biological fluids. Regarding implications of mitochondria in ALS, we have found that the FALS mutant hSOD1-G93A stabilises PINK1 in mitochondria and subsequently activates NFκB in neuronal cells. Sequential interaction between hSOD1 and NFκB impairs the proteosome proteolitic function promoting co-aggregation of SOD1 and PINK1 in these cells. These results add substantial mechanistic insight on the roles of mitochondria in classical ALS-associated neurodegenerative events, including aggregation of dysfuntional proteins in motoneurones. Following our study of mitochondria affectation in ALS, we have created and characterised a novel Drosophila model that expresses human SOD1-G93A in thoracic muscles under the genetic muscular promoter 24B. Flies expressing human SOD1-G93A in thoracic muscles successfully recapitulate FALS mitochondrial phenotype with several advantages in front of the current available rodent models for this FALS mutation. Taken together, the results generated in this thesis provide experimental evidence, further molecular comprehension and promise novel therapeutic approaches to the molecular mechanisms and neurodegenerative events associated with synaptic frailty and mitochondrial disfunction in FALS.

A list of genes have been identified to carry mutations causing familial ALS such as SOD1, TARDBP, C9orf72. But for sporadic ALS, which is 90% of all ALS cases, the underlying genetic variants are still largely unknown. There are multiple genome-wide association study (GWAS) for sporadic ALS, but usually a large number nominated SNP can hardly be replicated in larger cohort analysis. Also majority of GWAS SNP lie within noncoding region of genome, imposing a huge challenge to study their biological role in ALS pathology. With the rapid development of next-generation sequencing technology, we are able to sequence exome and whole-genome of a large number of ALS patients to search for novel genetic variants and their potential biological function. Here by analyzing exam data, we discovered two novel or extremely rare missense mutations of DPP6 from a Mestizo Mexican ALS family. We showed the two mutations could exert loss-of-function effect by affecting electrophysiological properties of Potassium channels as well as the membrane localization of DPP6. To our knowledge this is the first report of DPP6 nonsynonymous mutations in familial ALS patients. In addition, by analyzing whole-genome data, we discovered strong linkage disequilibrium between SNP rs12608932, a repeatedly significant ALS GWAS signal, and one polymorphic TGGA tetra-nucleotide tandem repeat, which is further flanked by large TGGA repetitive sequences. We also demonstrated rs12608932 risk allele is associated with reduced UNC13A expression level in human cerebellum and UNC13A knockout could lead to shorter survival in SOD1-G93A ALS mice. Thus the TGGA repeat might be the real underlying genetic variation that confer risk to sporadic ALS.

Swallowing and cough are two vital functions that are reflexive in nature and are related to each other in terms of shared neural and anatomical space. When a disorder impacts normal and effective swallowing and/or cough, the consequences can be life-threatening. Evaluation and treatment of swallowing and cough disorders can fall under the scope of practice of the speech-language pathologist and speech-language pathologists often are leading professionals. Furthermore, much of the current research on swallowing and cough is spearheaded by speech-language pathologists often working with a multi-disciplinary team. The focus of this dissertation is on the clinical evaluation of cough and swallowing, practice patterns of voluntary cough assessment during the evaluation of swallowing, and novel methods of evaluating acoustic voluntary cough waveforms in patients with and without swallowing impairment. The results will provide important information regarding the state of cough assessment tools for clinical swallowing evaluation, clinical practice patterns of voluntary cough assessment, and differences in acoustic cough signals between safe and unsafe swallowers in individuals with Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease of unclear aetiology, although the general consensus is of a multifactorial disease. The kynurenine pathway (KP), activated during neuroinflammation, is emerging as a possible contributory factor in ALS. The KP is the major route for tryptophan (TRP) catabolism. The intermediates generated can be either neurotoxic, such as quinolinic acid (QUIN), or neuroprotective, such as picolinic acid (PIC), an important endogenous metal chelator. The first and inducible enzyme is indoleamine 2,3-dioxygenase (IDO). As the extent of the involvement of the KP in ALS is unknown, the main aim of this thesis was to attempt to answer that question. The techniques used in this work include HPLC, GC/MS, RT-PCR, immunohistochemistry and immunocyctochemsitry. The main findings of this project are: (1) the complete KP is present in the mouse motor neuron cell line, NSC-34; (2) QUIN toxicity on NSC-34 cells may be ameliorated through the administration of NMDA antagonists, neuroprotective kynurenines, kynurenine inhibitor and QUIN monoclonal antibody; (3) in ALS patients, QUIN CSF and serum levels are significantly elevated, while PIC serum levels are significantly reduced; (4) ALS brain and spinal cord tissue show extensive microglia activation and positive immunoreactivity IDO and QUIN in spinal motor neurons and Betz cells in the motor cortex; and (5) kynurenine pathway inhibitor and analogue, R061-8048 and tranilast, are able to prolong the survival in the G93A SOD1 ALS transgenic mouse model. In conclusion, this study provide the first strong evidence for the involvement of the KP in ALS, and these data point to an inflammation-driven excitotoxic-chelation defective mechanism in ALS, which is amenable to KP analogue and inhibitor in ALS transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a late onset neuro-degenerative disorder characterized by highly selective death of large motor neurons in the cerebral cortex and spinal cord. A proportion of the familial cases (FALS) with autosomal dominant transmission was linked to chromosome 21q and the defective gene was shown to be the Cu/Zn superoxide dismutase gene (SOD1). SOD1 is a ubiquitously expressed cytoplasmic metalloenzyme catalyzing the dismutation of the superoxide free radical into hydrogen peroxide and molecular oxygen. We have screened our FALS patients for mutations in the SOD1 gene and found mutations in about 13% of the cases. All but one mutation were single base pair substitutions resulting in amino acid changes (i.e. missense mutations) predicted to produce structurally defective molecules, and some of which significantly reduced the SOD1 enzyme activity in lymphoblasts. We have also identified a two base pairs deletion, which introduces a premature stop codon at position 131 and is predicted to result in the translation of a truncated molecule.
It has been hypothesized that the pathology observed in FALS cases with SOD1 mutations is due to a gain of a new deleterious function of the mutant enzyme and not to a simple loss of dismutase activity. However the exact mechanism of SOD1 toxicity is still unknown and the specificity of the degenerating cell populations remains to be addressed. In this work, we investigated whether the damage seen in ALS with SOD1 mutations results from direct motor neuron toxicity. We have generated transgenic animals carrying a human SOD1 cDNA with the G37R mutation associated with FALS, driven by the neurofilament light chain promoter in order to specifically express the mutant protein in neuronal tissues. We show that transgenic animals express high levels of the human SOD1 protein in neuronal tissues, especially in the spinal cord where the motor neurons are concentrated, but develop no apparent motor deficit at up to 2 years of age. Our animal model suggests that neuron specific expression of mutant human SOD1 might not be sufficient for the development of the disease in mice, and hints towards the involvement of additional yet unidentified cell types/mechanisms.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease. Currently there is only one modestly beneficial pharmacological treatment, Riluzole, approved by the FDA. It has been documented that polyunsaturated fatty acid (PUFA) concentrations can affect the progression of neurodegenerative conditions however most interventions rely on nutritional supplementation and have limited long-term effectiveness. This thesis describes experiments using fenretinide, a synthetic retinoid capable of altering membrane PUFA concentrations, in a mouse model of ALS (SOD1(G93A)mice). Our treatment resulted in delayed onset, improved motor coordination, and increased life expectancy. Fenretinide also increased plasma levels of the ω-3 PUFA docosahexaenoic acid (DHA) while decreasing ω-6 PUFA arachidonic acid (AA) and products of lipid peroxidation malonyldialdehyde (MDA) and nitrotyrosine (NT). Spinal cord immunohistochemistry revealed a significant reduction in inflammation as assessed by the quantity of activated microglia and astrocytes. These results indicate that fenretinide represents a promising treatment strategy for ALS.
La Sclérose latérale amyotrophique (SLA) est la maladie affectant les neurones moteurs adultes la plus commune. Il n'existe qu'un seul traitement pharmacologique approuvé par la FDA ayant certains effets bénéfiques, soit le Riluzole. Il est par ailleurs documenté que des concentrations d'acides gras polyinsaturés (PUFA) peuvent affecter la progression d'un état neurodégénératif. Cependant, la plupart des interventions s'appuient sur des suppléments nutritifs et ont une efficacité à long terme plutôt limitée. Cette thèse décrit une série de traitements utilisant le fenretinide, un rétinoïde synthétique capable d'altérer la concentration de PUFA dans les membranes, dans un modèle de souris de SLA (souris SOD1(G93A)). Les traitements ont entrainé un retardement du déclenchement de la maladie avec une meilleure coordination motrice ainsi qu'une espérance de vie améliorée. Le fenretinide a également accrue les niveaux plasmatiques de l'acide docosahexaenoique tout en diminuant les niveaux d'arachidonate ainsi que les produits de peroxydation lipidiques tel que malonyldialdehye et nitrotyrosine. L'analyse immunohistochimique de la moelle épinière a révélé une réduction significative de l'inflammation déterminé par la quantité d'astrocytes et de microglies activés présentes. Ces résultats indiquent que le fenretinide représente un traitement prometteur contre la SLA.

Thesis: Ph. D. in Biomedical Engineering, Harvard-MIT Program in Health Sciences and Technology, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 213-227).
Progressive bulbar motor deterioration resulting from amyotrophic lateral sclerosis (ALS) leads to speech impairment. Despite the devastating consequences of speech impairment to life quality, few options are available to objectively assess speech motor involvement. The overarching goal of this research was to derive objective measures of speech acoustics that can be used to support clinical decision making. To achieve this goal, we obtained 121 speech samples from 33 patients with ALS who repeated the phrase "Buy Bobby a puppy" five times in succession. In total, 342 acoustic features were semi-automatically extracted from each speech recording. Pearson correlations were computed between each feature and three metrics of overall speech severity: sentence intelligibility, speaking rate, and communication efficiency. The findings were grounded within a physiologic framework where acoustic features were grouped into one of three domains that when combined, were hypothesized to broadly characterize articulatory performance: articulatory specification, articulatory coupling, and articulatory consistency. To obtain the most accurate prediction of ALS with the features we extracted, we compared two machine learning algorithms: linear regression and random forest. In shuffle-split cross-validation, the strongest mean Pearson correlations we obtained between actual and predicted intelligibility, speaking rate, and communication efficiency were 0.67, 0.74, and 0.77, respectively (SD=0.077, 0.050, and 0.059, respectively). Of the three domains, the specificity features were the most strongly associated with intelligibility impairments (mean r=0.68), and coupling was the most strongly associated with slower speaking rate (mean r=0.73). Specificity and coupling yielded similar performances in communication efficiency prediction. Other contributions of this thesis are that it is the first to implement a framework of dysarthric speech in terms of three domains: specification, coupling, and consistency; the first to validate automated formant tracking in dysarthric speech; and the first to perform an in-depth investigation into physiologically-inspired acoustic features that describe articulatory impairments of patients with ALS. Novel findings include the presence of abnormal formant coupling patterns, which may suggest greater tonguejaw coupling, in patients with more severe dysarthria due to ALS. Areas of future research involve further feature discovery, improved analysis methods, and a deeper understanding of relations to articulatory kinematics.
by Rachelle L. Horwitz-Martin.
Ph. D. in Biomedical Engineering

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (p. 224-236).
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with diagnostics and treatments that are ineffective at stopping the progression. This thesis examines new ways of both diagnosing and treating ALS, including 1) a gadolinium tetanus toxin C fragment (Gd-TTC) biomarker for axonal retrograde transport, 2) TTC-conjugated biodegradable nanoparticles, and 3) poly(glycerol-co-sebacate) acrylate (PGSA) and 3D scaffolds for human embryonic stem cell (hESC) and neuronal encapsulation.A Gd-TTC conjugate was developed and characterized that was shown to be highly visible under MRI and preserved the functionality of the native TTC protein in vitro. Live animal MRI imaging and immuno fluorescent staining of the spinal cord showed that the conjugate was transported to the central nervous system (CNS) and localized in motor neurons. H&E staining and biodistribution studies showed that GdTTC was well tolerated and bio available. Quantification of MRI and staining images showed that Gd-TTC was retrograde transported and that that this rate decreased during the disease progression of ALS in a transgenic mouse model, suggesting that Gd-TTC could be used as a biomarker for neurodegenerative diseases.TTC-conjugated nanoparticles were developed by synthesizing PLGA-PEG-biotin and using biotin binding proteins (avidin, streptavidin, and neutravidin) to specifically conjugate TTC to the nanoparticle surface. TTC nanoparticles were shown to selectively target neurons and not other cell types in vitro.
(cont.) Subsequent in vivo experiments showed that nanoparticles were well tolerated and that TTC was co-localized with neurons unilaterally, suggesting that TTC-conjugated nanoparticles may be a useful drug delivery system. Porous PGSA scaffolds were prepared and characterized by porosity, swelling, mass loss, toxicity and mechanical properties, and subsequently used to encapsulated hESC and neuroblastoma cells in vitro. Neuroblastoma cells proliferated and formed matrix fibrils, and fluorescent staining of undifferentiated hESCs showed the presence of all three germ layers. In vivo experiments showed that porous PGSA scaffolds were well-tolerated and promoted vascular ingrowths.
by Seth A. Townsend.
Ph.D.

Recent evidence has shown that non-neuronal cells within the CNS play a role in motoneuron degeneration in ALS. However, while it is accepted that various muscles are differentially affected within the disease, it is still unclear whether muscle has a causal role in ALS. In this Thesis, using a mouse model of ALS (mutant SOD1 transgenic mice), I examined muscle and NMJ pathology in two different muscle types, at various stages of disease progression. Using morphological and functional markers of innervation, I found that mSOD1- mediated pathology was present in fast-twitch muscles of mSOD1 mice prior to motoneuron degeneration. This occurred at a time when slow-twitch muscles remain innervated. This difference in the vulnerability of fast-twitch and slow-twitch muscles to disease was reflected in a differential heat shock response (HSR), with the slow-twitch soleus muscle displaying higher heat shock protein (hsp) levels over a longer duration of disease than the fast-twitch TA muscle. Equally, examination of the NOS response revealed a significant difference in nNOS expression between the fast-twitch TA and slow-twitch soleus muscles. Furthermore, fast-twitch muscles from mSOD1 mice also demonstrated greater vulnerability to cell stress such as glucose deprivation and caffeine stress in vitro. This Thesis also examined the effect of treatment with Arimoclomol, a pharmacological co-inducer of the HSR, on muscle and NMJ pathology. I found that treatment with was sufficient to decrease muscle pathology, augment the HSR and reduce mSOD1-meditated changes in nNOS expression. These findings support previous suggestions that targeting the periphery, in conjunction with the CNS, is an effective therapeutic strategy against ALS. Furthermore, that Arimoclomol’s previously reported beneficial effects are due, at least in part, to its effect in the periphery.

The susceptibility of MNs to injury in amyotrophic lateral sclerosis (ALS) may result from excitotoxicity and subsequent dysregulation of intracellular calcium homeostasis. Post-transcriptional editing of the AMPA and Kainate glutamate receptor (GLUR) subunit(s) may alter the calcium ion permeability of the receptor pore, leading to neurodegeneration. The aims of the study are (i) to investigate GLUR2, GLUR5 and GLUR6 RNA editing at Q/R site and its editing enzyme ADAR2 and ADAR3 in ALS/C9ORF72-positive and ALS/C9OR72-negative compared to non neurological controls, and (ii) to determine the aberrant expression of EAAT2 transcripts in the astrocytes of the spinal cord of ALS cases and controls. (iii) To characterise p-TDP43, p62, GLUR2, ADAR2, ADAR3 and GFAP and EAAT2 proteins in motor neurons of ALS/C9ORF72-postive and ALS/C9ORF72-negative cases and controls. (iv) To develop new methodology to look at different levels of the mRNA expression in GLUR2, GLUR5, GLUR6, ADAR2, ADAR3 and EAAT2 that might exist in ALS. RNA was extracted from MNs and cDNA was prepared followed by amplification of cDNA by PCR that was used to generate amplified GLUR2, GLUR5 and GLUR6. These products were digested to produce cuts in the internal site of GLUR mRNA and differentiate edited from non-edited GLUR by analysis using agarose gel electrophoresis and the Agilent Bioanalyser. Independently the mRNA expression of ADAR2, ADAR3 and EAAT2 alternative transcripts of normal and aberrant exon 4 and 9 were quantified by qPCR. p-TDP-43, p62, GLUR2, ADAR2, ADAR3 and GFAP and EAAT2 protein levels were assessed by immunohistochemistry. The mRNA expression of GLUR2, GLUR5, GLUR6, ADAR2, ADAR3 and EAAT2 was assessed by next generation sequencing It was demonstrated that GLUR2 was fully edited in the MNs of ALS/C9ORF72- positive, ALS/C9ORF72-negative cases and controls. GLUR5 was 39% edited in MNs of ALS/C9ORF72-positive, 57% edited in ALS/C9ORF72-negative cases and 30% edited in controls. GLUR6 editing in MNs was 86% editing in the ALS/C9ORF72- positive and 77% editing in ALS/C9ORF72-negative cases compared to 72% editing in controls. An elevated mRNA expression in EAAT2 aberrant and normal transcripts of exon 4 and exon 9 was shown, but did not reach tatistical significance. ADAR2 immunoreactivity was associated with the nuclei of MNs, in contrast to ADAR3 which was exclusively associated with the neuronal cytoplasm. No significant differences in the pattern or distribution of immunoreactivity was detected across all groups. Phosphorylated TDP-43+ and p62+ skein-like bodies and compact inclusions were significantly elevated in both ALS/C9ORF72-positive cases and ALS/C9ORF72- negative cases, compared to control subjects which exhibited neither p-TDP-43 nor p62 expression. No significant difference in the pattern of immunoreactivity of EAAT2 in ALS groups and controls. In conclusion, full editing of GLUR2 in dissected motor neurons isolated by LCM was confirmed in ALS/C9ORF72-positive and ALS/C9ORF72-negative and control individuals. Whereas partial editing has been identified in GLUR5 and GLUR6 receptors. The mRNA and protein expression of ADAR2 and ADAR3 on spinal cord have shown similar levels between tested groups. No significant difference in the mRNA and protein expression of EAAT2. MNs in ALS cases, both with and without C9ORF72 expansion expressed pathogenic inclusions of phosphorylated TDP-43 and p62, which are a hallmark for ALS. ADAR2 and ADAR3 have shown no differences in ALS groups compared to controls.

La grande variété de l'expérience émotionnelle peut-être interprétée en tant que fonction d'interactions entre des phénomènes cognitifs et émotionnels. Puisque certaines fonctions cognitives déclinent avec l'âge, parallèlement au control émotionnel sur une fine échelle, la relation entre le vieillissement et les états émotionnels induits a été examiné. Les sujets âgés, en comparaison avec des sujets jeunes, ont montré moins d'affects positifs et plus des émotions négatives. Puisque les conditions neuropathologiques, comme dans le cas des maladies dégénératives, présentent une influence considérable sur les processus cérébraux, une évaluation des fonctions cognitives et de la réponse émotionnelle a été entreprise chez des patients atteints de la Sclérose Latérale Amyotrophique (SLA), en comparaison à des sujets contrôles. L'évolution des paramètres cognitifs et émotionnels a été étudiée sur une période d'un an. Les patients (SLA) ont été significativement plus déprimés que les sujets contrôles, la dépression a augmenté au cours de la période de l'étude. Alors que pour les fonctions cognitives de mémoire (non-frontales), pas de changement a été observé, une performance déficitaire liée à des processus frontaux a été détecté, suggérant un léger déficit des fonctions cognitives. Ces déficits, à l'opposé des fonctions neuromusculaires et de la dépression, n'ont pas été aggravés au long de la période de l'étude. Les réponses émotionnelles, malgré une certaine variabilité qui peut-être le mieuxexpliquée en termes des profiles hétérogènes des patients, n'ont pas été significativement différentes entre les patients (SLA) et les sujets contrôles. Globalement, l'étude a mis en évidence un léger dysfonctionnement des processus cognitifs exécutifs chez les patients atteints de la SLA, qui évolue lentement, avec le temps. Contrairement, le profil émotionnel semble influencé par la maladie à un certain degré
The great variety of emotional experience can be explained as a function of emotion-cognition interactions. Given that some cognitive aspects decline with age, in parallel to finer degrees of emotional control, the relationship between age and emotionally induced feeling states was examined. Older subjects, compared with younger participants, showed less positive affects and more negative emotions. Since neuropathological conditions, such as in degenerative diseases, present a considerable impact on brain processes, an evaluation of cognitive function and emotional reactivity was undertaken in patients with ALS, compared to matched controls. The evolution of these parameters over a one year period was assessed. ALS patients were significantly more depressed than controls, and depression increased over the study period. While there was no observable change in memory and retrieval non-frontal cognitive functions, a performance deficit linked to frontal executive processing was detected suggesting a very mild defect in cognitive function. These deficits, unlike neuromuscular function and depression, did not aggravate over the period of the study. Emotional reactivity, in spite of some variability that can be best explained in terms of the heterogeneity of patients' profiles, did not differ significantly between ALS patients and controls. The study provides evidence for a mild defect in frontal cognitive processing in ALS patients, that evolves only slowly, if at all, with time. On the contrary, the profile of emotional processing in ALS seems to be affected to some extent