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Tjust, Anton. "Extraocular Muscles in Amyotrophic Lateral Sclerosis." Doctoral thesis, Umeå universitet, Anatomi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-129638.
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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.
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LOFFREDA, ALESSIA. "RNA Metabolism alteration in amyotrophic lateral sclerosis models." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/81488.
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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.
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AROSIO, ALESSANDRO. "Study of transcriptional alterations in Amyotrophic Lateral Sclerosis." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/94396.
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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.
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Rahmani, Kondori Nazanin. "Developing and testing therapies for Amyotrophic Lateral Sclerosis (ALS)." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/34340.
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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.
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Figueiredo, Joana Maria Serra de Oliveira Duarte. "The role of microRNAs in amyotrophic lateral sclerosis." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/7991.
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Dissertação para obtenção do Grau de Mestre em
Genética Molecular e BiomedicinaMicroRNAs (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.
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Jonsson, P. Andreas. "Superoxide dismutase 1 and amyotrophic lateral sclerosis." Doctoral thesis, Umeå : Medical Biosciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-611.
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Wootz, Hanna. "Amyotrophic Lateral Sclerosis – A Study in Transgenic Mice." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7342.
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Ekegren, Titti. "Transmethylation, Polyamines and Apoptosis in Amyotrophic Lateral Sclerosis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3952.
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Zetterström, Per. "Misfolded superoxide dismutase-1 in amyotrophic lateral sclerosis." Doctoral thesis, Umeå universitet, Klinisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-43898.
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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.
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Xu, Guang. "Identification of Novel Genetic Variations for Amyotrophic Lateral Sclerosis (ALS)." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/958.
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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.
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DIAMANTI, LUCA. "Identification of muscular MRI biomarkers in Amyotrophic Lateral Sclerosis (ALS)." Doctoral thesis, Università degli studi di Pavia, 2019. http://hdl.handle.net/11571/1301269.
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-Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease usually assumed to target motor neurons (MNs). However, evidence of involvement of other cells types, also outside the central nervous system, has challenged this neurocentric view of ALS, which may thus be defined a multi-systemic disease. Although muscle abnormalities in ALS are habitually considered secondary to MN damage, muscle, too, can be a primary target, with dying back degeneration of MNs occurring subsequently. In recent years, efforts to clarify the pathogenesis of ALS have focused on muscle tissue. Muscle magnetic resonance imaging (MRI), shown to be useful to define prognostic biomarkers of ALS in the G93A-SOD1 mouse model, is an established diagnostic tool in inherited and acquired neuromuscular disorders. Literature data on the role of muscle MRI in ALS are scarce and heterogeneous. To study the role of muscle MRI in the diagnosis and prognosis of ALS, we proposed two cross-sectional and one longitudinal studies. In the first study we compared qualitative T1-w images of hand, paraspinal and lower limb muscles in newly diagnosed ALS patients and in age-matched healthy controls (HCs) to look for evidence of muscular atrophy and remodelling (i.e. fatty substitution) and to relate the radiological findings to clinical and electromyographic (EMG) data. We concluded that muscle T1-w MRI can distinguish ALS patients from HCs for specific regions (i.e. legs). MRI abnormalities could be found in pauci-symptomatic spinal muscles in bulbar-onset patients (i.e. iliopsoas). Paraspinal and leg muscles MRI may be a useful diagnostic tool in early ALS. Form these preliminary results, we planned to conduct a longitudinal study in order to investigate more in deep the role of muscle through MRI measurements as a prognostic or predictive biomarker in ALS patients. In the second part we decided to focus the study on the role of paraspinal MRI in the diagnosis of ALS, and we qualitatively compared T1-w images in newly diagnosed ALS patients, age-matched healthy controls, patients affected by inflammatory myopathy and lumbar radiculopathy. We found that paraspinal T1-w MRI could help to distinguish spinal ALS patients from healthy and pathological controls. In particular, study of longissimus dorsi can play the role of diagnostic ALS biomarker. In the third study we compared quantitative images (6-point Dixon GRE and multi-echo TSE T2-w obtaining Water T2 and Fat Fraction values) of lower limb muscles in ALS patients at different timepoints to look for evidence of disease progression and to relate the radiological findings to clinical data. We found that ALS patients showed acute alterations in muscles more than controls at baseline but not at following timepoints. Moreover, ALS patients and controls had no difference in fat fraction at baseline, but the difference is significant during the follow up. In conclusion, MRI pattern traces the pathological process of ALS and muscle MRI can be useful as prognostic biomarker.
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Menon, Parvathi. "The role of the corticomotorneurons in pathogenesis of amyotrophic lateral sclerosis." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/11609.
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Amyotrophic lateral sclerosis (ALS) is a progressive, degenerative disease of the motor system clinically defined by the presence of upper and lower motor neuron (LMN) signs. The site of onset of pathophysiology within the motor system in ALS remains unresolved and this thesis examines the role of the corticomotor neuron in the pathogenesis of ALS. The diagnostic utility of the split-hand sign in ALS involving preferential wasting of the ‘thenar’ group of intrinsic hand muscles namely the abductor pollicis brevis (APB) and first dorsal interosseous (FDI) was established by recording the split-hand index (SI) which was noted to reliably differentiate ALS from mimic neuromuscular disorders. The cortical and axonal excitability characteristics of the ‘thenar’ muscles namely the APB and FDI was compared with the hypothenar abductor digiti minimi (ADM) with threshold tracking transcranial magnetic stimulation (TMS) studies revealing cortical hyperexcitability to be a feature of ALS pronounced over the ‘thenar’ muscles while axonal hyperexcitability while a feature of ALS, did not selectively affect the prominently wasted ‘thenar’ muscles. Cortical hyperexcitability was also noted to precede the development of lower motor neuron dysfunction in a clinically and neurophysiologically normal APB muscle. The selective vulnerability of muscles in ALS was further defined by the split hand plus sign with a greater degree of cortical hyperexcitability over the preferentially wasted APB muscle in ALS patients when compared with a similarly innervated and relatively preserved flexor pollicis longus (FPL) muscle. In summary, corticomotorneuronal hyperexcitability as a marker of corticomotorneuronal dysfunction predominates over the muscles which are preferentially wasted in ALS and precedes evidence of lower motor neuron loss. The findings presented in this thesis support the primacy of the corticomotor neuron in the pathogenesis of the split hand phenomenon and suggest a mechanism for the pathogenesis of ALS.
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Morimoto, Emiko. "The Role of Microglia in Amyotrophic Lateral Sclerosis: Analysis of MicroRNAs." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10155.
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Amyotrophic lateral sclerosis (ALS) is a progressive adult onset neurodegenerative disease characterized by selective death of the upper and lower motor neurons of the brain and spinal cord. Neuromuscular synapses are lost leading to paralysis and ultimately death. Non-neuronal cells, such as astrocytes, oligodendrocytes, and microglia, have been shown to contribute to ALS disease progression in mouse models. Microglia, the innate immune cells of the central nervous system, have been shown to be activated in ALS and contribute to disease progression. Hundreds of mRNAs have shown to be dysregulated in a variety of ALS cell types and tissues, including total spinal cord, acutely isolated microglia, and in vitro differentiated motor neurons. These mRNAs can be regulated post-transcriptionally by microRNAs (miRNAs), which are small endogenous non-coding RNAs with important regulatory roles in a wide range of cellular processes. This dissertation examines the contribution of miRNAs to ALS disease progression in microglia. I acutely isolated primary microglia from the spinal cords of transgenic mice overexpressing human wild type (WT) SOD1 and human G93A SOD1. I used small RNA sequencing to profile the miRNAs that are expressed during disease progression, and identified miRNAs that are differentially expressed. I confirmed these results by quantitative PCR and examined the expression changes of predicted targets in a microglia RNA-seq dataset. Here I show that miRNAs are dysregulated in acutely isolated microglia from SOD1 G93A transgenic mice, and that miR-155, a pro-inflammatory miRNA, and miR-210, a hypoxia-inducible miRNA, are significantly upregulated during disease progression. In addition, miR-1198-5p,
miR-182, miR-503, and miR-668 are also dysregulated, and predicted mRNA targets of all six of these miRNAs are differentially expressed during disease progression. To my knowledge, this is the first analysis of miRNA expression in microglia during ALS disease progression. This work contributes to the understanding of the contribution of a non-neuronal cell type to ALS disease progression and serves as a paradigm for studies in other non-neuronal cell types, such as astrocytes and oligodendrocytes, and other ALS mouse models.
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Kasi, Patrick K. "Characterization of motor unit discharge rate in patients with amyotrophic lateral sclerosis." Worcester, Mass. : Worcester Polytechnic Institute, 2009. http://www.wpi.edu/Pubs/ETD/Available/etd-050409-062647/.
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Highlander, Morgan Michelle. "Electroceutical Therapy in Amyotrophic Lateral Sclerosis: A Novel Preliminary Study." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1530099548144113.
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Jacoby, Adam M. "The Role of Nitric Oxide and Nitroxidative Stress in Amyotrophic Lateral Sclerosis." Ohio University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1273265480.
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Forsberg, Karin. "Misfolded superoxide dismutase-1 in sporadic and familial Amyotrophic Lateral Sclerosis." Doctoral thesis, Umeå universitet, Patologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-47550.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features. The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder. Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates. In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.
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Bergemalm, Daniel. "Mutant superoxide dismutase-1-caused pathogenesis in amyotrophic lateral sclerosis." Doctoral thesis, Umeå : Umeå university, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-31116.
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Wagner, Karin Nicole. "Amyotrophic Lateral Sclerosis and Genetic Testing: A Perspective from the ALS Community." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1459354988.
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Pettit, Lewis David. "White matter integrity, executive dysfunction, and processing speed in amyotrophic lateral sclerosis." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9843.
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Cognitive impairment in amyotrophic lateral sclerosis (ALS) is characterized by deficits on tests of executive functions however the contribution of processing speed is unknown. By contrast, multiple sclerosis (MS) is a disorder in which slowed processing speed is regarded as the core deficit, however, methodology is often confounded by tasks which depend on motor speed. MRI studies have revealed multi-system cerebral involvement in ALS, with evidence of reduced white matter volume and integrity in predominantly frontotemporal regions. The current study had two aims. Firstly, to investigate whether cognitive impairments in ALS and MS are due to executive dysfunction or slowed processing speed, independent of motor dysfunction. Secondly, to investigate the relationship between specific cognitive impairments and the integrity of distinct white matter tracts in ALS. Twenty-nine ALS patients, twenty-five MS patients, and matched healthy control groups were administered a dual task paradigm and processing speed tasks in which stimulus presentation times were manipulated. In addition background measures of executive functioning, working memory, verbal memory, and language were administered. White matter integrity was investigated using region-of-interest (ROI) and tract based spatial statistics (TBSS) analyses of diffusion MRI data. ALS patients did not show impairments in tests of processing speed, but deficits were revealed in the dual task, as well as background tests of executive functioning, working memory, and verbal memory. MS patients also exhibited deficits in the dual task as well as background tests of executive functioning, working memory, and verbal memory. However, in contrast to ALS patients, a processing speed deficit was also observed in MS. ROI analyses revealed significant differences in fractional anisotropy (FA) and mean diffusivity (
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DURANTI, ELISA. "Study of DUX4 as a novel biomarker in Amyotrophic Lateral Sclerosis." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2023. https://hdl.handle.net/10281/403046.
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La sclerosi laterale amiotrofica (SLA) è una malattia degenerativa di eziologia sconosciuta caratterizzata dalla compromissione dei motoneuroni superiori e inferiori. I fattori di rischio associati all'insorgenza e alla progressione della SLA rimangono sconosciuti. L'ipotesi più accreditata sull'eziopatogenesi della SLA prevede una concomitanza tra fattori genetici e ambientali che possono causare o contribuire allo sviluppo di forme familiari e sporadiche di SLA. Simile ad altre malattie neurodegenerative, la SLA è caratterizzata da un accumulo anomalo di proteine insolubili nel citoplasma dei motoneuroni in degenerazione. In particolare, nella SLA sporadica, si ritiene che l'aggregazione della proteina TDP-43 svolga un ruolo chiave nella degenerazione dei motoneuroni.
In questo studio, abbiamo studiato un nuovo possibile biomarcatore per la SLA: double homeobox 4 (DUX4). Infatti, in uno studio di Homma del 2015, è stato dimostrato che la overespressione della proteina DUX4 nei mioblasti di pazienti con distrofia facioscapolomerale (FSHD) può contribuire all'aggregazione di TDP-43. Pertanto, nella prima parte del nostro studio, abbiamo valutato la possibile espressione di DUX4 e la sua correlazione con l'espressione di TDP-43 nelle cellule mononucleate del sangue periferico (PBMC) di pazienti con SLA. Successivamente, valutato l’espressione di DUX4 anche in campioni di post mortem, fibroblasti e sui motoneuroni derivati da iPSCs. In conclusione, è stata eseguita una trasfezione di DUX4 in linee cellulari di neuroblastoma umano e abbiamo valutato l'espressione di TDP-43. Nell'ultima parte, abbiamo valutato gli effetti dei farmaci approvati dalla FDA per il trattamento della SLA, Edaravone e Riluzolo, sull'espressione di DUX4.
Inizialmente, abbiamo analizzato le cellule mononucleate del sangue periferico (PBMC) ottenute da 46 pazienti con SLA e 26 controlli possibili alterazioni nell'espressione di DUX4. Abbiamo riscontrato un aumento significativo dell'mRNA di DUX4 e dei livelli proteici nei PBMC dei pazienti rispetto ai controlli. Secondo i risultati precedenti del nostro gruppo (Arosio et al., 2020), abbiamo successivamente confermato un aumento dei livelli di proteina TDP-43 nelle PBMC della SLA, nonché un aumento delle forme troncate di TDP-35 e TDP-25 e abbiamo verificato una correlazione positiva tra i livelli di proteine DUX4 e TDP-43, TDP-35 e TDP-25 nei campioni di SLA. Inoltre, le nostre analisi di immunofluorescenza delle PBMC ALS hanno mostrato la co-localizzazione di DUX4-TDP-43 nella regione perinucleare con una tendenza ad aggregarsi. Anche gli esperimenti eseguiti sui fibroblasti SLA e sui motoneuroni derivati da iPSCs con l'espansione patologica di C9ORF72 hanno indicato un'analoga maggiore espressione di DUX4. Infine, sono stati condotti studi di immunoistochimica su campioni post mortem di corteccia e midollo spinale ottenuti da pazienti con SLA. I motoneuroni superiori nei campioni di corteccia motoria della SLA hanno mostrato un'immunoreattività a DUX chiaramente rilevabile, mentre quasi nessun segnale era presente nei campioni di controllo. DUX4 invece non è espresso nei campioni di midollo spinale, indipendentemente dallo stato patologico. Infine, abbiamo eseguito in linee cellulari di neuroblastoma umano una trasfezione DUX4 e, dimostrando che la upregolazione di DUX4 dopo la trasfezione ha indotto un aumento della frazione proteica insolubile TDP-43.
Ad oggi, nessuna terapia può curare la SLA, sebbene ci siano due farmaci, Riluzolo ed Edaravone, approvati dalla Food and Drug Administration (FDA). Pertanto, nella parte finale di questo studio, abbiamo studiato i possibili effetti di questi due farmaci sull'espressione di DUX4 in linee cellulari di neuroblastoma umano con mutazione SOD1 wild type o G93A. I nostri risultati hanno mostrato che i farmaci hanno ridotto l'espressione di DUX4 in condizioni basali e in seguito allo stress ossidativo indotto dal trattamento con H2O2Amyotrophic lateral sclerosis (ALS) is a degenerative neuromuscular disease of unknown etiology characterized by the impairment of both upper and lower motor neurons. The risk factors associated with ALS onset and progression remain unknown. The most accepted hypothesis on the etiopathogenesis of ALS foresees a concomitance between genetic and environmental factors that can cause or contribute to the development of familial and sporadic forms of ALS. Similar to other neurodegenerative diseases, ALS is characterized by an abnormal accumulation of insoluble proteins in the cytoplasm of degenerating motor neurons. In particular, in sporadic ALS, the aggregation of TDP-43 protein is thought to play a key role in the degeneration of motor neurons. In this study, we investigated a novel possible biomarker for ALS: double homeobox 4 (DUX4). In fact, in a study by Homma and colleagues in 2015, it was shown that the overexpression of the DUX4 protein in myoblasts from patients with facioscapulohumeral muscular dystrophy (FSHD) can contribute to the accumulation and aggregation of TDP-43. Therefore, in the first part of our study, we evaluated the possible expression of DUX4 and its correlation with TDP-43 expression in peripheral blood mononuclear cells (PBMCs) from patients with ALS. Subsequently, we performed in neuroblastoma cell lines a DUX4 transfection and we evaluated the expression of TDP-43 before and after treatment. In the last part of this study, we assessed the possible effects of the two drugs approved by the FDA for the treatment of ALS, Edaravone and Riluzole, on DUX4 expression. We analyzed peripheral blood mononuclear cells (PBMC) obtained from 46 patients with ALS and 26 controls for possible alterations in DUX4 expression. We found a significant (about threefold) increase in DUX4 mRNA and protein levels in PBMCs from patients compared to controls. According to previous results from our group (Arosio et al., 2020), we subsequently confirmed an increase in TDP-43 protein levels in ALS PBMCs, as well as an increase in the truncated forms of TDP-35 and TDP-25, and we showed a positive correlation between DUX4 and TDP-43, TDP-35, and TDP-25 protein levels in ALS samples. In addition, our immunofluorescence analyses of ALS PBMCs showed DUX4-TDP-43 colocalization in the perinuclear region with a tendency to aggregate. Also experiments performed on fibroblasts ALS and iPSCs-derived motor neurons carrying C9ORF72 pathological expansion indicated an analogous increased DUX4 expression. Finally, immunohistochemistry studies were performed on cortex and spinal cord postmortem samples obtained by ALS patients. Upper motor neurons in ALS motor cortex samples showed a clearly detectable DUX-like immunoreactivity, while almost no signal was present in control samples. On the other hand, DUX4 was not expressed in spinal cord samples, regardless of the pathological status. Finally, we performed in a human neuroblastoma cell lines (SH-SY5Y) a DUX4 transfection and, showing that DUX4 upregulation after transfection induced an increase in the TDP-43 insoluble protein fraction. To date, no therapy can cure ALS, although there are two drugs, Riluzole and Edaravone, have been approved by the Food and Drug Administration (FDA) for treatment. Thus, in the final part of this study, we investigated the possible effects of these two drugs on DUX4 mRNA and protein expression in neuroblastoma cell lines transfected with human SOD1 wild type or G93A mutation. Our results showed that Edaravone and Riluzole reduced DUX4 expression in basal conditions and following oxidative stress induced by H2O2 treatment.
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Lee, Rena J. "Study of trace and minor elements in ALS (amyotrophic lateral sclerosis) patients." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/36492.
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Ababneh, Nidaa. "Modelling of amyotrophic lateral sclerosis (ALS) using induced pluripotent stem cells (iPSC)." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:b0e48523-2acc-4c1e-83a5-79696cbaf042.
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The hexanucleotide repeat expansion (HRE) mutation within C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several hypotheses have been proposed for how the mutation contributes to pathogenicity, including the loss of C9orf72 gene function, RNA-mediate toxicity and the formation of toxic dipeptides by repeat-associated non-ATG (RAN) translation. Patient-specific iPSCs provide a promising tool for the study of the cellular and molecular mechanisms of human diseases in relevant cell types and discovering potential therapies. The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-mediated homology directed repair (HDR) system represents an attractive approach for disease modelling and development of therapeutic strategies. In this thesis, iPSCs derived from ALS/FTD patient carrying the HRE mutation were generated and subsequently gene edited to remove a massive repeat expansion from the patient cells and replace it with the wild-type size of the repeats using HDR and a plasmid donor template. The successful genotypic correction of the mutation resulted in the normalization of the C9orf72 gene promoter methylation level and the gene variants RNA expression level. Removal of the mutation also resulted in abolition of sense and antisense RNA foci formation and reduction of DPRs accumulation. Furthermore, the repeat size correction also rescued the susceptibility of cells to Glutamate excitotoxicity, decreased the apoptotic cell death and stress granules formation under the baseline and stress conditions. This work provides a proof-of-principle that removal of the HRE can rescue ALS disease phenotypes and provides an evidence that HRE mutation is an attractive target for therapeutic strategies and drug screening, to block the underlying disease mechanisms.
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Gallart, Palau Xavier Ramon. "Synaptic frailty and mitochondrial dysfunction in familial amyotrophic lateral sclerosis." Doctoral thesis, Universitat de Lleida, 2016. http://hdl.handle.net/10803/386410.
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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.
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Bergmann, Friederike. "Mitochondrial metabolism in hypoglossal motoneurons from mouse implications for amyotrophic lateral sclerosis (ALS) /." Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=975113429.
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Frakes, Ashley E. "The Role of Neuroinflammation in the Pathogenesis of Amyotrophic Lateral Sclerosis." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417649954.
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Burrell, Kathleen Ann. "The role of pathogenic SOD1 mutations in neuronal cell death in amyotrophic lateral sclerosis." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313992.
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Forrest, Stuart Gordon. "Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8096.
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The human VAMP-associated protein B (hVAPB) has been shown to cause a range of motor neurodegenerative diseases, including amyotrophic lateral sclerosis 8 (ALS8) and spinal muscular atrophy (SMA). However, the molecular mechanisms underlying VAPB-induced neurodegeneration remain elusive. We sought to address this question by identifying VAPB interacting proteins, which may be affected by the disease causative mutations. Using a combination of biochemical and genetic approaches in Drosophila, we confirmed the evolutionarily conserved phosphoinositide phosphatase Sac1 (Suppressor of Actin 1), as a DVAP binding partner and showed that the two proteins colocalise in the endoplasmic reticulum. We also show that DVAP function is required to maintain normal levels of phosphoinositides (PIs) and that downregulation of either Sac1 or DVAP at the larval neuromuscular junction (NMJ) affects a number of synaptic processes, including axonal transport, synaptic growth, microtubule integrity and localisation of several postsynaptic components. We found that double knock down of DVAP and Sac1 induces no further increase in the severity of the mutant phenotypes when compared to either single mutant alone. This, together with the similarity in mutant phenotypes, indicates that the two genes function in a common pathway. In flies carrying the ALS8 mutation (DVAP-P58S), we observed reduced viability, locomotion defects and early death in surviving adults, closely matching the phenotypes of both DVAP and Sac1 downregulation. Additionally, transgenic expression of DVAP-P58S in the motor system elicits synaptic defects similar to those of either Sac1 or DVAP loss-of-function, including an increase in the levels of PtdIns-4-Phosphate (PI4P), the substrate of Sac1. Consistent with these observations, we found that Sac1 is sequestered into DVAP-P58S mediated aggregates and that downregulation of PI4P in neurons rescues the neurodegenerative and the synaptic phenotypes associated with DVAP-P58S transgenic expression. Together our data unveil a previously unknown function for Sac1 in neurodegeneration and synaptic function, as well as provide evidence for a dominant negative mechanism for phosphoinositide-mediated ALS8 pathogenesis. We also highlight a causative role for increased PI4P levels in VAPB-P56S induced neurodegeneration.
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RUSCONI, MICHELA. "Activation state and functionality of dendritic cells from peripheral blood of amyotrophic lateral sclerosis patients." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/153236.
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Molti dati pubblicati sottolineano l’importanza dell’infiammazione per la neurodegenerazione nella sclerosi laterale amiotrofica (SLA) con un aumento del reclutamento al midollo spinale dei monociti periferici, delle cellule dendritiche (DCs) e delle cellule T in modelli murini e nell’uomo. Ad oggi non ci sono dati circa lo stato funzionale delle DCs nel sangue periferico dei pazienti SLA.
Lo scopo dello studio è esaminare le DCs circolanti in una coorte di pazienti SLA prendendo in considerazione la loro storia clinica, per capire come queste cellule contribuiscano alla progressione della patologia. Per fare questo abbiamo analizzato la frequenza di espressione delle molecole costimolatorie, migratorie e delle MHC delle DCs CD1c+ e abbiamo indagato la capacità di DCs purificate di produrre spontaneamente e in risposta ad agonisti dei TLR (lipopolisaccaride LPS) citochine infiammatorie.
Abbiamo incluso nello studio 72 pazienti SLA, 47 donatori sani e 25 pazienti affetti da disordini neurologici non correlati con la SLA, stratificati per sesso ed età. Il numero di DCs circolanti ed il loro fenotipo sono stati analizzati con analisi citofluorimetriche. Abbiamo trovato che i pazienti SLA hanno un numero minore di DCs circolanti rispetto a donatori sani e che queste DCs esprimono più bassi livelli di ntegrina CD62L. Questa integrina è necessaria per il reclutamento dei leucociti agli organi linfoidi secondari o ai siti infiammatori quindi la nostra osservazione conferma il fatto che nei pazienti SLA le DCs sono reclutate attivamente al sistema nervoso centrale con un meccanismo che coinvolge, presumibilmente, CD62L. Abbiamo poi analizzato la produzione spontanea o indotta da LPS di citochine infiammatorie quali TNFα, IL1β, IL6, IL8, IL10 eCCL2. Abbiamo individuato una sottopopolazione di pazienti con una produzione spontanea di IL8 più alta che mostrano anche una più alta efficienza di secrezione di CCL2. Nonostante non sia stato possibile dimostrare una correlazione tra questa più alta efficienza di secrezione di citochine e la progressione della patologia, alti livelli di CCL2 sono presenti nel midollo spinale di topi SOD, un modello murino per una sottoclasse di pazienti SLA, e in alcuni pazienti. Questa osservazione suggerisce che delle semplici analisi del sangue periferico potrebbero essere sufficienti ad identificare un sottogruppo di pazienti. Abbiamo inoltre analizzato la correlazione tra i livelli di ogni singolo citochina, prima e dopo l’esposizione ad LPS, ed alcuni parametri di malattia. Abbiamo così evidenziato una correlazione inversa tra l’intervallo di tempo tra l’insorgenza e la diagnosi e i livelli di ΔIL6, suggerendo che l’efficienza di produzione di IL6 possa accelerare le fasi iniziale della patologia. In conclusione possiamo dire che le DCs sono il subset cellulare maggiormente reclutato al sistema nervoso centrale. Nonostante la maggior parte delle DCs attivate migrino al sistema nervoso centrale, alcune differenze posso essere osservate a livello del sangue periferico. Sulla base dei nostri risultati le analisi sul sangue periferico potrebbero essere utili a stratificare i pazienti dividendo chi ha un’alta risposta infiammatoria da chi invece non ha alterazioni. Data l’elevata eterogeneità della SLA non è stato possibile, per il momento, osservare correlazioni significative con i parametri di malattia, anche se analisi più raffinate basate su specifici criteri potrebbero essere informative su alcuni particolari aspetti della patologia. L’elevato livello di espressione di CD62L da parte delle DCs periferiche suggerisce che questa molecola possa essere un target per un trattamento in vivo. A questo proposito abbiamo in programma di mettere a punto uno studio preclinico in topi SOD per verificare se un trattamento con un anticorpo bloccante CD62L possa interferire con la progressione della patologia.Several published data highlight the importance of inflammation for neurodegeneration in Amyotrophic lateral sclerosis (ALS) with an increased spinal cord recruitment of peripheral proinflammatory monocytes, dendritic cells (DCs) and T cells found in patients and animal models. To date no clear data are available regarding the functional state of DCs in peripheral blood of ALS patients.The aim of the present study was to examine circulating DCs in a large cohort of ALS patients taking into account their clinical phase in order to lay the basis to understand how these cells contribute to disease progression. To do this, we performed ex vivo analyses of the frequency and expression of costimulatory, MHC and migratory mole¬cules of CD1c+ DC subsets and we investigated the capacity of purified DCs to spontaneously produce inflammatory cytokines and to respond to the TLR agonist, lipopolysaccharide (LPS). We enrolled 72 ALS patients, 47 healthy donors and 25 Patients affected by neurological disorders unrelated with ALS stratified for age and sex. DC numbers and their phenotype were investigated by cytofluorimetric analyses. We found that ALS patients have much lower number of circulating DCs (identified as CD1c+ and CD19-) compared with healthy donor, and their DCs show an increased expression of the integrin CD62L. Since this integrin is required for the recruitment of leukocytes to secondary lymphoid organs or to inflammatory sites, these observations confirmed that in ALS patients DCs are actively recruited in the central nervous system with a mechanism presumably involving the CD62L molecule. We then analysed the spontaneous o LPS-induced production of inflammatory cytokines such as TNFα, IL1β, IL6, IL8, IL10 and CCL2. We noticed a subpopulation of ALS patients with a higher spontaneous and LPS-induced IL8 production. These patients, interestingly, also showed higher efficiency of CCL2 secretion. Although we could not define a correlation between the higher efficiency of these inflammatory cytokine production and disease progression, high levels of CCL2 have been shown in the spinal cord of SOD mice, a mouse model of a subclass of ALS disease, and in some ALS patients. According to our results, DCs can be a source of CCL2 in the spinal cord in a subpopulation of ALS patients. This observation suggests that a simple peripheral blood analysis can be sufficient to identify subgroups of ALS patients. We, thus, analysed the correlation between the levels of any single cytokines in ALS patients before and after LPS exposure and some disease parameters.We observed a significant inverse correlation between the time from onset to diagnosis and the ΔIL6 levels, suggesting that an increased efficiency of IL-6 production in ALS patients may accelerate the initial phases of the disease.In conclusion, DCs are one of the major cell subset recruited to the central nervous system at least in some ALS patients. Although the majority of activated DCs may migrate to the central nervous system, some differences are still observable in the peripheral blood. Based on our results, peripheral blood DC analyses can be useful to stratify patients in those that have a high inflammatory response versus those that do not show an altered inflammatory pathway.Given the high heterogeneity of ALS disease we could not observe for the moment significant correlations with disease parameters, nevertheless a more refined analysis based on specific criteria is likely to be informative on some particular disease aspects.The high levels of CD62L expression by peripheral blood DCs suggest that this molecule could be a possible target for in vivo treatment. To this regard, we are planning to perform a preclinical study in SOD mice to verify if a treatment with a blocking anti-CD62L antibody could interfere with disease progression.
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Torres, Cabestany Pascual. "Cell Stress and RNA Splicing in Amyotrophic Lateral Sclerosis: Novel Opportunities for Therapeutic Development." Doctoral thesis, Universitat de Lleida, 2021. http://hdl.handle.net/10803/671440.
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L'ELA és una malaltia neurodegenerativa multifactorial. Atès que actualment és una malaltia incurable sense una causa coneguda, els esforços per descobrir noves dianes terapèutiques estan ben justificats. L'evidència disponible indica que la fisiopatologia de l'ELA pot estar composta parcialment d'interaccions entre alteracions de lípids, estrès oxidatiu, deslocalització de TDP-43 i metabolisme de l'ARN. Per explorar l'impacte de la deslocalització de TDP-43, quantifiquem per primera vegada l’splicing críptic derivat de la disfunció de TDP-43 en teixit humà, models cel·lulars i ratolins. Els exons críptics són candidats a biomarcadors en l'ELA, i els quantifiquem en altres patologies relacionades amb alteracions de TDP-43, com la demència tipus Alzheimer-LATE. Un dels ARNm controlats per TDP-43 codifica per ATG4B, una proteïna essencial en la formació de la maquinària de membrana de l'autofàgia. D'altra banda, les membranes de el sistema nerviós central estan enriquides en àcids grassos poliinsaturats (PUFA), molt sensibles a l'estrès oxidatiu. Les publicacions anteriors de el grup demostren una disminució en l'àcid docosahexaenoic (DHA) -un PUFA- a la medul·la espinal dels donants d'ELA. El DHA és un regulador crucial de la inflamació i s'ha relacionat amb malalties neurodegeneratives relacionades amb l'edat. Aquesta tesi mostra que la intervenció dietètica pot augmentar eficaçment el contingut de DHA de la medul·la espinal mitjançant l'ús d'una dieta enriquida amb DHA, que té un efecte beneficiós en la supervivència dels ratolins transgènics mascles. Aquesta millora es va associar amb una disminució dels marcadors inflamatoris i de dany de l'ADN. A causa de que s'observa un dany en l'ADN similar i un perfil d'alliberament de citocines en el perfil secretor associat a la senescència (SASP) en la neuroinflamació, també examinem el paper potencial de la senescència en l'ELA. Vam detectar nivells augmentats de marcadors SASP i signes de senescència cel·lular (expressió augmentada de p16-INK i p21) en etapes presimptomàtiques i simptomàtiques en la medul·la espinal dels ratolins hSOD1-G93A. No obstant això, el tractament senolític amb Navitoclax (un inhibidor de Bcl-2) no va oferir cap benefici en la supervivència dels ratolins ni una disminució dels nivells d'expressió de gens de senescència cel·lular i marcadors SASP. També vam demostrar que l'splicing críptic augmenta en ratolins hSOD1-G93A en l'etapa final i es correlaciona amb el marcador de senescència p16-INK. Finalment, per millorar el potencial de transferència, vam explorar les alteracions de l'splicing críptic i canvis metabolòmics com a potencials biomarcadors perifèrics. Desafortunadament, els exons críptics mesurats en teixit nerviós no s'expressen en cèl·lules mononuclears de sang perifèrica. No obstant això, les anàlisis del metaboloma plaquetari van revelar canvis significatius en els enfocaments de casos-controls i de pronòstic. En conclusió, aquesta tesi destaca les noves troballes relacionades amb possibles intervencions dietètiques i senolítics, i proposa nous biomarcadors metabolòmics i transcriptòmics.La ELA es una enfermedad neurodegenerativa multifactorial. Dado que actualmente es una enfermedad incurable sin una causa conocida, los esfuerzos para descubrir nuevas dianas terapéuticas están bien justificados. La evidencia disponible indica que la fisiopatología de la ELA puede estar compuesta parcialmente de interacciones entre alteraciones de lípidos, estrés oxidativo, deslocalización de TDP-43 y metabolismo del ARN. Para explorar el impacto de la deslocalización de TDP-43, cuantificamos por primera vez el splicing críptico derivado de la disfunción de TDP-43 en tejido humano, modelos celulares y ratones. Los exones crípticos son candidatos a biomarcadores en la ELA, y los cuantificamos en otras patologías relacionadas con alteraciones de TDP-43, como la demencia tipo Alzheimer-LATE. Uno de los ARNm controlados por TDP-43 codifica para ATG4B, una proteína esencial en la formación de la maquinaria de membrana de la autofagia. Por otro lado, las membranas del sistema nervioso central están enriquecidas en ácidos grasos poliinsaturados (PUFA), muy sensibles al estrés oxidativo. Las publicaciones anteriores del grupo demuestran una disminución en el ácido docosahexaenoico (DHA) -un PUFA- en la médula espinal de los donantes de ELA. El DHA es un regulador crucial de la inflamación y se ha relacionado con enfermedades neurodegenerativas relacionadas con la edad. Esta tesis muestra que la intervención dietética puede aumentar eficazmente el contenido de DHA de la médula espinal mediante el uso de una dieta enriquecida con DHA, que tiene un efecto beneficioso en la supervivencia de los ratones transgénicos machos. Esta mejora se asoció con una disminución de los marcadores inflamatorios y de daño del ADN. Debido a que se observa un daño en el ADN similar y un perfil de liberación de citocinas en el perfil secretor asociado a la senescencia (SASP) en la neuroinflamación, también examinamos el papel potencial de la senescencia en la ELA. Detectamos niveles aumentados de marcadores SASP y signos de senescencia celular (expresión aumentada de p16-INK y p21) en etapas presintomáticas y sintomáticas en la médula espinal de los ratones hSOD1-G93A. Sin embargo, el tratamiento senolítico con Navitoclax (un inhibidor de Bcl-2) no ofreció ningún beneficio en la supervivencia de los ratones ni una disminución de los niveles de expresión de genes de senescencia celular y marcadores SASP. También demostramos que el splicing críptico aumenta en ratones hSOD1-G93A en la etapa final y se correlaciona con el marcador de senescencia p16-INK. Por último, para mejorar el potencial de transferencia, exploramos las alteraciones del splicing críptico y cambios metabolómicos como potenciales biomarcadores periféricos. Desafortunadamente, los exones crípticos medidos en tejido nervioso no se expresan en células mononucleares de sangre periférica. No obstante, los análisis del metaboloma plaquetario revelaron cambios significativos en los enfoques de casos-controles y de pronóstico. En conclusión, esta tesis destaca los hallazgos novedosos relacionados con posibles intervenciones dietéticas y senolíticos, y propone nuevos biomarcadores metabolómicos y transcriptómicos.
ALS is a multifactorial neurodegenerative disease. As it is currently an incurable disease without a known cause, the efforts in discovering novel targets are well justified. Available evidence indicates that ALS pathophysiology can be partially comprised of interactions between lipid alterations, oxidative stress, TDP-43 mislocalization, and RNA metabolism. In order to explore the impact of TDP-43 mislocalization, we quantified for the first time the cryptic exon splicing derived from TDP-43 dysfunction in human tissue, cell models, and mice. Cryptic exons are candidate biomarkers in ALS, and we quantified them in other pathologies related to TDP-43 disturbances, such as the Alzheimer-LATE type dementia. One of the TDP-43 controlled mRNAs is codifying for ATG4B, an essential protein at the build-up of autophagy's membranal machinery. On the other hand, the central nervous system membranes are enriched in polyunsaturated fatty acids (PUFAs), which are very sensitive to oxidative stress. The group's previous publications indicated a decrease in docosahexaenoic acid (DHA) -a PUFA- in ALS donors' spinal cord. DHA is a crucial regulator of inflammation and has been implicated in age-related neurodegenerative diseases. This thesis shows that dietary intervention can effectively increase DHA content of the spinal cord by using a DHA enriched diet, which has a beneficial effect on male survival time. This improvement was associated with decreased inflammatory and DNA damage markers. Because a similar DNA damage and cytokine release profile are seen in senescence-associated secretory profile (SASP) in neuroinflammation, we also examined the potential role of senescence in ALS. We detected increased levels of SASP markers and signs of cell senescence (increased expression of p16-INK and p21) in pre- and symptomatic stages in the hSOD1-G93A ALS spinal cord. However, a senolytic treatment with Navitoclax (a Bcl-2 inhibitor) did not offer any benefit in mice survival nor a decreased level of cell senescence and SASP markers. We also demonstrate that cryptic exon splicing is increased in the hSOD1-G93A ALS model at the end-stage, and it is correlated with senescence marker p16-INK mRNA. Finally, to enhance transference potential, we explored both metabolomic and cryptic exon splicing alterations in peripheral biomarkers. Unfortunately, cryptic exons measured in nervous tissue are not expressed in peripheral blood mononuclear cells. Notwithstanding, platelet metabolome analyses revealed significant changes in case-control and prognostic approaches. Globally, this thesis highlights novel findings related to potential dietary interventions, open the door for new therapeutic agents such as senolytic and propose new metabolomics and transcriptomic biomarkers.
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Klepek, Holly N. "Genetic testing in Amyotrophic Lateral Sclerosis: A Survey of ALS Clinicians and Commercial Testing Laboratories." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523829253204339.
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Palmieri, Arianna. "A neuropsychological and cognitive insight in amyotrophic lateral sclerosis / motor neuron disease." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425586.
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A neuropsychological and cognitive insight into motor neuron disease/amyotrophic lateral sclerosis (MND/ALS)
1) We evaluated cognitive functioning in 128 MND patients (vs 113 healthy controls), with a comprehensive neuropsychological battery. Dysfunctions were significantly present in 40% of cases (mainly for executive functioning and short-term memory); 7% of them showed clear frontal or fronto-temporal dementia (chapter 2).
2) Nine ALS patients (vs 10 healthy controls) underwent the fMRI study: significant differences were displayed in lateralized activation between patients and controls during the attentional task, both for unpleasant and for neutral word stimuli (statistically greater activation in right middle frontal gyrus for patients, greater activation for frontal, parietal and cerebellar areas in controls) and lower activation was shown in patients in the posterior cingulate during mnesic recall, for both unpleasant and neutral stimuli (chapter 3).
3) We validated an Italian version of a specific quality of life questionnaire for MND/ALS: the ALSAQ-40, psychometric reliability in terms of internal reliability, construct validity, test-retest reliability and face validity were evaluated on the basis of 76 patients’ responses. Correlation with functional and clinical measures are discussed in the text (chapter 4).
5) Similarly, we validated an Italian version of the ELQ, (a questionnaire for detecting emotional lability) on 41 MND patients and 39 respective caregivers (vs 39 healthy controls and 39 pseudo-caregivers). The lack of correlation with neuropsychological profile and correlations with psychopathological indices, both in patients and in caregivers, are described in the text (chapter 5).
5) The Rorschach test (according to Exner’s guidelines) was administred to 21 ALS patients with early onset, 21 with onset longer than two years and 21 myasthenic controls. Among the numerous findings obtained, suicidal ideation was significantly more present in the ALS group with a recent diagnosis compared to those with a more remote one (chapter 6).
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Proudfoot, Malcolm. "Cortical neurophysiology of ALS." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:5b8673f7-8eb2-4bf2-b3b8-d901fa134007.
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The experiments described in this thesis aimed to investigate the neurophysiological consequences, at the cortical level, of the neurodegenerative condition, amyotrophic lateral sclerosis (ALS). A principle tenet of this study was that ALS is, first and foremost, a disorder of the cortical motor system, the precise pathological mechanisms of which remain incompletely understood. Furthermore, the degree to which neurodegeneration can be evidenced before the onset of symptoms is thus far uncertain, and the optimal means by which to measure therapeutic response has yet to be determined. Chapter 1 introduces relevant key concepts in ALS and briefly summarises three studies completed in the early phases of pursuit for the above degree. These studies respectively considered presmyptomatic cellular ALS pathology, quantitation of disease progression and eyetracking assessment of cognitive dysfunction. Chapter 2 describes magnetoencephalography, the investigative technology utilised in the subsequent experimental chapter. In chapter 3, the effects of ALS on movement related modulation of neuronal oscillations are determined. An excessive peri-movement desynchronisation and delayed post-movement rebound was described. Functional connectivity between cortical regions at rest is appraised in chapter 4. ALS appeared to result in quite striking increases in functional connectivity, in keeping with the fMRI literature and in support of diminished intracortical inhibitory influences. The functional communication from the motor cortices is directly considered during active motor performance in chapter 5. ALS related reductions in beta-band coherence were noted in both corticospinal and inter- hemispheric communication. In conclusion, the results demonstrated considerable support for proposed excitotoxic disease mechanisms and were in alignment with reported findings in other neurodegenerative diseases. Finally, a pilot study by which the neural mechanisms for cognitive impairment in ALS are explored via antisaccade performance is described. While underpowered, the experimental design showed promise for future application.
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Schmidt, Eric J. "Investigation of a Misfolded, Destabilized Profilin-1 Species as a Toxic Molecule in ALS Pathogenesis." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1043.
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Dominant mutations in profilin-1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by motor neuron loss, paralysis, and death from respiratory failure. Our lab recently demonstrated that PFN1 mutant proteins are destabilized—they unfold at milder conditions during thermal and chemical denaturation. Furthermore, we and others have shown that mutant PFN1 is more prone to misfold and aggregate. This misfolding alters PFN1’s protein-protein interactions, as demonstrated by an affinity purification-mass spectrometry screen. While ALS-associated mutants do not show loss of interaction, several have altered interactions with several formin family proteins, a group of proteins that interacts with profilins to regulate actin polymerization. These perturbations in profilin-formin interaction result in changes in actin metabolism, as shown by stress fiber formation in a HeLa model and neurite outgrowth in an iPSC-derived neuron model. Additionally, one mutant shows increased actin filament survival time in a microfluidic experiment, indicative of tighter binding in the actin-profilin-formin complex at the growing end of a filament. Misfolding and aggregation also puts additional stress on the cell’s proteostasis pathways. A cell culture model shows that misfolded Pfn1 is processed primarily by the proteasome, with modest contributions from autophagy. Together, this evidence provides additional support for two theories of Pfn1 ALS pathogenesis: disruptions in cytoskeletal function and proteostatic stress.
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Teyssou, Elisa. "Analyses génétiques et fonctionnelles de nouveaux gènes incriminés dans la Sclérose Latérale Amyotrophique (SLA) Genetic analysis of matrin 3 gene in French amyotrophic lateral sclerosis patients and frontotemporal lobar degeneration with amyotrophic lateral sclerosis patients Genetic analysis of CHCHD10 in French familial amyotrophic lateral sclerosis patients." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066738.
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La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative fatale caractérisée par la dégénérescence des motoneurones centraux et périphériques. Elle est le plus souvent sporadique (SALS, 90% des cas), tandis que les formes familiales (FALS) représentent 10% des patients. Une vingtaine de gènes liés à la SLA ont été identifiés et sont responsables de 70% des FALS et 10% des SALS. Le but de ce projet était d’étudier la contribution de 6 gènes rares dans une large cohorte de patients français atteints de SLA et d’étudier les conséquences fonctionnelles de certains variants identifiés. La première partie de ce projet a consisté à réaliser l’analyse génétique des gènes MATR3, CHCHD10, SS18L1, SQSTM1, UBQLN2 et PFN1. Aucun variant causal ne fut identifié pour les 2 premiers gènes alors que 2 nouveaux variants possiblement pathogènes ont été identifiés dans le gène SS18L1, 4 mutations pour SQSTM1, 5 dans UBQLN2 et 2 mutations déjà répertoriées dans le gène PFN1. Cette analyse génétique a permis de souligner un chevauchement génétique entre SLA et maladie de Paget pour SQSTM1 et entre SLA et Paraplégie Spastique pour UBQLN2. La deuxième partie de ce projet a consisté en l’étude de la pathogénicité de certains variants que nous avons identifiés dans les gènes SQSTM1, UBQLN2 et PFN1, par l’analyse (i) des inclusions dans les tissus post mortem de patients, (ii) de l’expression et de la dégradation protéique dans des lymphoblastes issus de patients SLA et/ou (iii) des conséquences cellulaires après surexpression in vitro et in vivo. Ces analyses ont montré, concernant SQSTM1, qui est impliquée dans la formation des autophagosomes, que les mutations perturbaient l’agrégation protéique, que les mutations dans le gène UBQLN2 altéraient la dégradation lysosomale et la liaison de la protéine avec HSP70 et que celles dans PFN1 dérégulaient la voie de l’autophagie alternative et la mitophagie. Notre travail a permis (i) d’évaluer la contribution chez les patients français atteints de SLA de plusieurs gènes incriminés dans la SLA, (i) d’élargir le spectre génétique commun à la SLA et à d’autres pathologies et (iii) de mettre en avant la pertinence de l’implication des voies de dégradation protéique, notamment l’autophagie, dans la pathogénèse de la maladieThe fatal Amyotrophic Lateral Sclerosis (ALS) motor neuron disease is characterized by the degeneration of upper and lower motor neurons. Most ALS cases are sporadic (SALS) whereas ~10% are familial (FALS). A growing number of genes has been identified in ALS and represent 70% of FALS and 10% of SALS. The aims of this project were to analyze the contribution of 6 rare genes in a large population of French ALS patients and to study the pathogenic impact of some identified variants.The first part of this work was dedicated to the genetic analysis of MATR3, CHCHD10, SS18L1, SQSTM1, UBQLN2 and PFN1 genes. No causing variants were identified for MATR3 and CHCHD10 while 2 new variants, probably pathogenic, were identified for SS18L1, as well as 4 mutations for SQSTM1, 5 for UBQLN2 and 2 already reported mutations for PFN1. These analyses also highlighted a genetic overlap between ALS and other diseases: the Paget disease of bone for SQSTM1 and spastic paraplegia for UBQLN2. The second part of this work was to study the pathogenicity of some of the mutations identified in SQSTM1, UBQLN2 and PFN1 genes using analyses of (i) inclusions in ALS patient post-mortem tissue, (ii) protein expression and degradation pathways in patient lymphoblasts and/or (iii) cellular consequences after in vitro and in vivo overexpression. Our results showed prominent aggregation of mutant SQSTM1 (involved in autophagosomes formation), impaired lysosomal degradation and disrupted protein binding to HSP70 for mutant UBQLN2 and deregulated alternative autophagy and mitophagy pathways for mutant PFN1. Our results (i) precised the contribution of several genes in French ALS patients, (i) documented the genetic overlap between ALS and other diseases and (iii) highlighted the role of protein degradation pathways, especially autophagy, in the pathogenesis of ALS
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Camara, Mafalda Dias de Medeiros Vale da. "Coherence and phase locking disruption in electromyograms of patients with amyotrophic lateral sclerosis." Master's thesis, Faculdade de Ciências e Tecnologia, 2013. http://hdl.handle.net/10362/10950.
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Dissertação para obtenção do Grau de Mestre em
Engenharia BiomédicaIn motor neuron disease, the aim of therapy is to prevent or slow neuronal degeneration and early diagnosis is thus essential. Hypothesising that beta-band (15-30 Hz) is a measure of pathways integrity as shown in literature, coherence and PLF could be used as an electrophysiological indicator of upper and lower neuron integrity in patients with ALS. Before further analysis, synthetic EMG signals were computed to verify the used algorithm. Coherence and PLF analyses were performed for instants of steady contraction from contra and ipsilateral acquisitions. Ipsilateral acquisitions were performed for one member of each group and results present significant differences between both groups. Contrarily, contralateral acquisitions were performed on 6 members of each group and results present no significant differences. PLF analysis was computed for ipsilateral acquisitions and, similarly to coherence, results show significant differences between both groups. PLF was also analysed for contralateral acquisitions, and results show no significant differences within groups, as expected since no coherence was found for the same acquisitions. So, while control subjects present no neuronal or muscular problems and therefore higher synchrony and coherence for beta-band frequencies, patients with ALS do not present synchronism or coherence in any frequency, specially for beta-band. All results allowed to conclude that contralateral coherence is not a good measure of corticospinal pathways integrity. However, ipsilateral acquisitions show promising results and it is possible to affirm that ipsilateral measurements may reflect neuronal degeneration. For future work is suggested a deeper analysis of PLF, that appear to have potential as a quantitative test of upper and lower neuron integrity related to ALS.
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Watermeyer, Tamlyn Julie. "Emotional processing and social cognition in Amyotrophic Lateral Sclerosis (ALS)/Motor Neuron Disease (MND)." Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/emotional-processing-and-social-cognition-in-amyotrophic-lateral-sclerosis-als--motor-neuron-disease-mnd(e3552e39-2127-40a8-8035-357b66edd75d).html.
Full textAbstract:
Amyotrophic Lateral Sclerosis (ALS) is a debilitating and life–limiting neurodegenerative disorder that causes progressive muscle atrophy and spasticity. A small proportion of ALS patients experience co–morbid Frontotemporal Dementia (FTD). Milder cognitive–behavioural changes have been noted in ALS patients without dementia. In these patients, deficits in executive functioning, language, memory and behaviour have been documented. Recently, changes to emotional processing and social cognition (EMOSOC) in ALS have also been reported, albeit with inconsistent findings. The primary aims of the current thesis were i) to delineate the nature and extent of changes in EMOSOC in ALS and ii) to determine the relationship between such changes and interindividual differences in mood, behaviour, personality, empathy and ALS–related executive dysfunction. The results of the study indicate a profile of predominant executive dysfunction, with relative sparing of EMOSOC in non–demented ALS patients. However, the ALS patients did show impaired performance on a task requiring the attribution of thoughts and feelings to characters from cartoons and vignettes. ALS patients’ performance on EMOSOC tasks was predicted by their performance on tests of executive function, above and beyond mood, behaviour, personality and empathy variables. As a secondary aim, the impact of patients’ cognitive and behavioural changes on ALS caregivers’ outcomes (mood, perceived strain, burden and marital satisfaction) were examined. The data indicated patients’ behavioural dysfunction and functional impairment as key predictors of caregivers’ outcomes. Exploratory analyses revealed differences between patients’ and caregivers’ perceptions of patients’ personality, empathy and behaviour; these differences were associated with caregiver outcomes. In summary, the current thesis characterises the profile of EMOSOC changes in non–demented ALS and highlights the role of ALS–related executive dysfunction in these changes. It also assesses the relative impact of patients’ disease, cognitive and behavioural changes on ALS caregivers.
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GIANNINI, MARTA. "Regulatory role of TDP-43 on R-loop formation in Amyotrophic Lateral Sclerosis (ALS)." Doctoral thesis, Università degli studi di Pavia, 2019. http://hdl.handle.net/11571/1301290.
Full textAbstract:
Gli R-loops sono costituiti da un ibrido RNA-DNA più un filamento di DNA dislocato (ssDNA) e sono naturalmente coinvolti in processi cellulari come la trascrizione del DNA, la replicazione mitocondriale e nucleare del DNA o il cambio di classe delle immunoglobuline (Ig). Il loro sviluppo è favorito da specifiche aree del genoma arricchito in cluster GC, detti GC skew, tipici dei promotori, grazie alla stabilità termodinamica della struttura ibrida RNA-DNA ad una frequenza maggiore nei siti trascritti di loci genomici e quindi possono agire come intermedi cotranscrizionali. In alcune condizioni patologiche, i fattori fisiologici che impediscono la formazione di R-loop sono compromessi e quindi la formazione persistente di R-loop può diventare un risultato rischioso con effetti deleteri sull'integrità del genoma. La conseguenza del loro accumulo è fortemente correlata all'instabilità genomica che favorisce lo sviluppo di riarrangiamenti di sequenze ectopiche di DNA omologhi, iperricombinazione del DNA, riarrangiamenti cromosomici o, quando abortivo, a perdita cromosomica. È stato dimostrato che gli R-loop sono associati a malattie autoimmuni neuroinfiammatorie o a disturbi neurodegenerativi mentre la loro relazione con la Sclerosi Laterale Amiotrofica (SLA) è ancora in fase di studio. La SLA è la più comune malattia motoneuronale ad insorgenza adulta ed è caratterizzata dalla progressiva perdita dei motoneuroni superiori e inferiori del midollo spinale, del tronco cerebrale e della corteccia motoria, con conseguente debolezza muscolare ed eventuale insufficienza respiratoria. Nonostante la natura multifattoriale di questa patologia, la ribonucleoproteina TDP-43 è stata recentemente identificata come la principale proteina patologica nella SLA sporadica. Nelle inclusioni citoplasmatiche positive al TDP-43, la proteina è anormalmente fosforilata, ubiquitinata e troncata con conseguente accumulo di frammenti C-terminali (CTF25 e CTF35), che evitano che TDP-43 non eserciti la sua funzione fisiologica nucleare legata al metabolismo dell'RNA, compresi splicing, traduzione e trasporto. In alternativa, le mutazioni del TDP-43 (1-2% dei casi), che si raggruppano principalmente nella regione che codifica la regione a bassa complessità (LC) ricca di glicina del C-terminus, possono essere neurotossiche attraverso un nuovo meccanismo di guadagno di funzione, sviluppando caratteristiche che possono essere direttamente connesse con la patologia, come l'aumento della propensione all'aggregazione e dell'emivita, la localizzazione subcellulare alterata e le interazioni proteina-proteina. E' stato dimostrato il coinvolgimento di TDP-43 anche nella risposta al danno al DNA (DDR) come componente critico della via di riparazione della rottura del DNA a doppio filamento mediata dal pathway NHEJ. La colocalizzazione di TDP-43 con RNA polimerasi II attiva nei siti di danno al DNA insieme alla proteina di riparazione del danno al DNA, BRCA1, dimostra la sua partecipazione alla prevenzione o alla riparazione del danno al DNA associato con la formazione di R-loop. Inoltre le mutazione nella regione C-terminale sono associate alla perdita della sua importante funzione nucleare e sono nota per essere associate ad R-loops eccessivamente stabili, meccanismi di trascrizione in stallo e DNA stabile aperto. Quindi, a causa di alterazioni o mutazioni di questa ribonucleoproteina, i processi cellulari coinvolti nella modulazione dell'RNA e nella regolazione che cooperano con la risoluzione o la soppressione dei fattori ibridi RNA-DNA sono influenzati, portando a accumulo di R-loops. A sua volta, questo meccanismo aumenta la quantità di trscritti aberranti, che promuovono ulteriormente la neurodegenerazione cellulare. Lo scopo di questo progetto si è basato sull'indagine del ruolo regolatorio del TDP-43 nella formazione di R-loop utilizzando SH-SY5Y, HeLa e LCLs linee cellulari linfoblastoidi derivanti da pazienti affetti da SLA.R-loops are three-strand nucleic acid structures constituted by an RNA-DNA hybrid plus a displaced DNA strand (ssDNA) that are naturally involved in cellular processes such as DNA transcription, mitochondrial and nuclear DNA replication or immunoglobulin (Ig) class switching. Their development is favored by specific areas of genome enriched in GC clusters, called GC skew, such as promoter regions, thanks to the thermodynamic stability of RNA-DNA hybrid structure. A certain number of reports suggest that R-loops are formed at a higher frequency in transcribed sites of genomic loci and can act as cotranscriptional intermediates. In some pathological conditions, physiological factors that prevent formation of R-loops are impaired and hence R-loops persistent formation can become a risky outcome with deleterious effects on genome integrity. The consequence of their accumulation is strongly related to genomic instability enhancing the development of ectopic homologous DNA sequences rearrangements, DNA hyper recombination, chromosomal rearrangements or, when abortive, to chromosome loss. It was shown that R-loops are associated with neuroinflammatory autoimmune disease such as Aicardi-Goutière Syndrome (AGS) or with neurodegenerative disorders such as fragile X-associated tremor/ataxia syndrome, while their relation with Amyotrophic Lateral Sclerosis (ALS) is still under study. ALS is the most common adult-onset motor neuron disease, and is characterized by the progressive loss of upper and lower motor neurons from the spinal cord, brain stem, and motor cortex, leading to muscle weakness and eventual respiratory failure. Despite the multifactorial nature of this pathology, TDP-43 was recently identified as the major pathological protein in sporadic ALS. In TDP-43-positive cytoplasmic inclusions, the protein is abnormally phosphorylated, ubiquitinated and truncated resulting in the accumulation of 25-kDa and 35-kDa C-terminal fragment (CTF25 and CTF35), that avoid full length TDP-43 from exerting its nuclear physiological function related to RNA metabolism, including splicing, translation, and transport. Alternatively, TDP-43 mutations (1-2% cases), which mainly cluster in the region encoding the C-terminus glycine-rich lowcomplexity region (LC), may be neurotoxic through a novel gain of-function mechanism, developing features that can be directly connected with the pathology, such as increased aggregation propensity and half-life, altered sub-cellular localization and proteinprotein interactions. Recently, emerging evidence in literature suggests the involvement of TDP-43 even in DNA damage response (DDR) as a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. Colocalization of TDP-43 with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, demonstrates its participation in the prevention or repair of R loop associated DNA damage. As a consequence, TDP-43 depletion leads to increased sensitivity to various forms of transcription-associated DNA damage such as R-loops- 2 associated damage and the mutation in C-terminus glycine-rich low-complexity region, LC domains associated with the loss of its important nuclear function, is known to be associated with excessively stable R-loops, stalled transcription machinery and stable open DNA. So due to impairments or mutations of this ribonucleoprotein, cellular processes involved in RNA modulation and regulation cooperating with resolving or suppressing RNA-DNA hybrid factors are affected, leading to accumulation of R-loops. In turn, this mechanism increases the amount of aberrant transcripts, which further promotes cellular neurodegeneration. The aim of this project was based on the investigation of the regulatory role of TDP-43 in R-loops formation using different in vitro cellular model such as SH-SY5Y, HeLa cervical cancer cells and LCLs derived from ALS patients.
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Kebe, Aicha R. "Characterization of Mechanisms for Suppressing Toxicity of ALS-Associated Protein FUS." Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright15669286599466.
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Kim, Soo Hyun. "Gene therapy demonstrates that muscle is not a primary target for non-cell autonomous toxicity in familial ALS." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1164829314.
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Sanhueza, Cubillos Mario Andrés. "Identification of novel genes interacting with DVAP, the causative gene of ALS8 in humans." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/21080.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease caused by the death of motor neurons leading to paralysis. Mechanisms underlying the pathogenesis of the disease remain unknown but with the identification of causative genes from ALS patients, some processes have been linked to the disease. One of these genes is VAPB, a highly conserved protein involved in lipid transfer, vesicle metabolism and synaptic morphology. We modeled in Drosophila the disease-linked P56S mutation (DVAP-P58S) and observed with the expression of this allele neurodegeneration in the eye and loss of motor performance. These phenotypes provide an excellent opportunity to use fly’s genetics to find novel genetic interactors of DVAP and understand ALS pathomechanism. Therefore, we carried out a large scale genetic screen by crossing the ALS model with a collection of P-element overexpression lines. After the analysis of 1183 lines, we obtained 71 modifier lines that suppress DVAP-induced neurodegeneration and 14 lines that enhance this phenotype, decreasing furthermore the eye size and viability of the offspring. To confirm that the effect of modifier lines was caused by a specific gene, we validated them with independent alleles of those genes. Using different sources, we were able to confirm the effect of 63 of the 85 modifiers, providing a strong confirmation of their effect. When we studied the effect of the modifier genes co-expressed with DVAP-P58S in the nervous system, we detected that 46 lines presented the same modifying effect in adult viability and 58 in the motor performance of the adult offspring. Considering the stronger readouts, we obtained 42 genes as novel high confidence DVAP genetic interactors. To understand furthermore the way they are affecting DVAP neurodegeneration, we carried out a series of bioinformatic analyses using Drosophila and human databases. Lipid droplets, vesicle metabolism and cell proliferation appear as the most important categories found in the screen, all processes conserved when analysed with human orthologs of the modifiers. Further characterisation of the endocytosis-linked modifier Rab5 and the predicted DVAP-interactors Rab7 and Rab11, showed that the suppression effect is not only confirmed in vivo but is also conserved in human tissue from ALS patients. These data validate our genetic screen and at the same time open novel opportunities to understand ALS mechanisms and find possible therapeutic targets.
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Toro, Gabriela. "Gene Therapy for Amyotrophic Lateral Sclerosis: An AAV Mediated RNAi Approach for Autosomal Dominant C9ORF72 Associated ALS." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1020.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation, and 4) Disrupted nucleocytoplasmic trafficking. Due to lack of treatments for this disease, we have pursued an AAV-RNAi dependent gene therapy approach, using an artificial microRNA (amiR) packaged in a recombinant adeno-associated virus (rAAV). After validating our in vitro results, we advanced to in vivo experiments using transgenic mice that recapitulate the major histopathological features seen in human ALS/FTD patients. Adult and neonate mice were injected through clinically relevant routes and our results indicate that AAV9-mediated amiR silencing not only reduced mRNA and protein levels of C9ORF72 but also the expansion derived toxic GP dipeptides. Although our amiR is not targeting the expansion itself but exon 3, we illustrate here that the evident dipeptide decrease is achievable due to the presence of aberrant transcripts in the cytoplasm containing miss-spliced Intron-HRE-C9ORF72 species. These encouraging results have led to the continued testing of this treatment as a therapeutic option for C9ORF72 - ALS patients.
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Graffmo, Karin Sixtensdotter. "Of mice and men : SOD1 associated human amyotrophic lateral sclerosis and transgenic mouse models." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1376.
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Crockford, Christopher James. "Neuropsychological functioning across the ALS disease course and its assessment." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33135.
Full textAbstract:
Amyotrophic Lateral Sclerosis (ALS) is a rapid and fatal neurodegenerative disease marked by progressive muscle weakness and wasting. Approximately 50% of people with ALS experience changes in cognition and behaviour. Previous research has been mixed as to whether cognition declines over the course of ALS, or whether it is related to proxies of disease progression (e.g., functional disability scales). However, this research has suffered from limitations including the use of inappropriate measures of cognition, imprecise measures of disease progression, high attrition, practice effects, and biased analytic approaches. Fortunately, recent advances in clinical assessment have provided accurate measures of neuropsychological functioning and disease progression, namely, the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and the King's Clinical Disease Staging. The present study aims to utilise recent advances in ALS disease metrics to overcome previous limitations and explore the evolution of cognitive and behavioural dysfunction over the course of ALS. Specifically, the aims of the present project are to 1) develop alternate forms of the ECAS to accommodate repeated longitudinal assessment; 2) examine how cognition and behaviour relate to clinical disease stages in ALS; 3) evaluate how cognitive and behavioural symptoms evolve over the course of the disease in ALS; and 4) explore clinicians' attitudes toward cognitive and behavioural screening in ALS. To achieve Aim 1, two new versions of the ECAS (ECAS-B and ECAS-C) were developed and administered to a group of age, education, and gender matched controls to that of the original ECAS-A validation study. Results demonstrate that the alternate forms of the ECAS (B and C) were equivalent to the original ECAS-A, reducing practice effects and possessing excellent inter-rater reliability. The ECAS forms were administered longitudinally to a separate group of healthy controls. Over an interval of 4 months, the ECAS-A-B-C showed no evidence of practice effects and excellent test-retest reliability validating their utility in the longitudinal monitoring of cognition in ALS. The ECAS forms were then used in an international multi-centre clinical sample of 161 ALS patients and 80 matched controls to achieve Aim 2. Patients were grouped into their King's Clinical Disease Stage at time of testing. Analysis revealed a significant cross-sectional relationship between disease stage and ALS-Specific cognitive functions, driven by a decline in verbal fluency performance. A significant relationship was also observed between disease stage and behavioural features. By end-stage disease 80% of patients demonstrated neuropsychological impairment. Participants were followed up longitudinally to explore the progression of cognitive and behavioural symptoms. Latent Growth Curve models of the ECAS subdomains (utilising the alternate versions) demonstrated a significant decline in ALS Specific cognitive, but not behavioural, functioning over time. This decline was explained by advancing disease stage, the presence of the C9orf72 repeat expansion, and years of education. Rate of change in ALS Non-Specific functions was dependent on baseline performance. Visuospatial functions and perseveration declined at similar rates and were distinct from language, fluency, apathy, and disinhibited behaviour. Cluster analysis of patients revealed a three-cluster solution with one group demonstrating no significant decline, a second group with mild cognitive and behavioural decline, and a third group with more severe neuropsychological decline. When data was restructured by diseases stage, rather than time, longitudinal results were similar to cross-sectional findings. To examine clinician's attitudes to cognitive and behavioural screening. Fourteen Health Care Professionals (HCP) working in ALS (Neurologists, Psychologists, and Clinical Care Specialists) were interviewed. Thematic analysis revealed that HCPs recognised the importance of cognitive and behavioural screening in ALS, but that it is not common practice. Important barriers to screening were reported including other members of staff, a lack of resources, and issues concerning patients and their families. Participants suggested increasing training and psychology input, and making screening a standardised protocol to all patients may alleviate these barriers. Cognition and behaviour are critically related to advancing disease stage, both cross-sectionally and longitudinally. Declining cognitive and behavioural symptoms has important implications for clinical practice, caregiver impact, and end-of-life decision making. However, clinicians report that cognitive and behavioural screening is not common practice and that significant barriers exist. The newly developed alternate forms of the ECAS provide an accurate, effective, and clinically useful means of monitoring cognitive function over the course of the disease in ALS.
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Bourefis, Annis-Rayan. "Novel FUS and CHCHD10 models to investigate pathogenic mechanisms in Amyotrophic Lateral Sclerosis." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS177.
Full textAbstract:
La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dévastatrice causée par la dégénérescence progressive des motoneurones (MNs) supérieurs et inférieurs menant à une faiblesse et une atrophie musculaire qui progresse jusqu’à la paralysie. Deux gènes majeurs identifiés chez les patients SLA sont le gène FUS (FUSed in sarcoma), impliqué dans le métabolisme de l’ARN, et CHCHD10, qui joue un rôle dans la stabilité des mitochondries. Ces deux gènes ont été étudiés à travers différents modèles, de petits modèles invertébrés aux biopsies de patients. Cependant, les différents traits phénotypiques observés sont complexes et parfois controversés. L’objectif de cette thèse est de fournir de nouvelles informations sur l’implication de ces deux gènes dans la SLA à travers l’utilisation de nouveaux modèles. Pour étudier les mécanismes pathologiques induits par FUS et CHCHD10, nous avons généré et caractérisé deux nouveaux modèles de poisson-zèbres présentant une mutation non-sens des orthologues de ces gènes, et nous avons mis en évidence différents traits phénotypiques propres à la SLA. Nous avons montré, pour FUS, une espérance de vie réduite, une locomotion altérée, des axones moteurs aberrants, une jonction neuromusculaire (JNM) désorganisée, une altération des muscles et mitochondries, ainsi que des changements moléculaires. Ces résultats montrent que la perte de fonction de fus est responsable de l’apparition de signes pathologiques distaux au niveau de la JNM, indiquant une neuronopathie en « dying-back », dans laquelle les traits pathologiques de la SLA commencent au niveau de la JNM et progressent vers les corps cellulaires des MNsAmyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder caused by progressive degeneration of upper and lower motor neurons (MNs), with a very rapid clinical course. It leads to muscle weakness and atrophy progressing to paralysis, with respiratory failure being the major cause of death within years following clinical diagnosis. Two major genes mutated in ALS patients are the RNA-binding protein FUS (FUSed in sarcoma), implicated in RNA metabolism, and coiled-coil-helix-coiled-coil-helix domain 10 (CHCHD10), which plays a role in mitochondria stability. Both these genes have been investigated through different model systems, from small invertebrate models to patient biopsies. However, the major phenotypic features obtained in these models are complex and often controversial. The objective of this work is to provide new insights on the implication of these genes in ALS through the use of new models.To investigate the pathogenic mechanisms induced by FUS and CHCHD10, we generated and characterized two novel stable non-sense mutant zebrafish models for the orthologues of these genes and highlighted several ALS phenotypic features. We demonstrated, for the FUS model but not for CHCHD10, reduced lifespan, locomotor disabilities, aberrant motor axons, disorganized neuromuscular junction (NMJ), muscle and mitochondrial alteration, as well as molecular changes. These findings indicate that loss of fus expression is responsible for the occurrence of distal pathological signs at the NMJ, thus supporting a “dying-back” neuronopathy, in which early disease hallmarks start at the level of the NMJ and progress towards MN cell bodies
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Cone, Alan J. "Fission Yeast as a Model Organism for FUS-Dependent Cytotoxicity in Amyotrophic Lateral Sclerosis." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1470750088.
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Patel, Anjali. "An Integrated Proteomic Approach for Mapping the ALS-linked TDP-43 Interactome." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42687.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which an RNA-binding protein, TDP-43, mislocalizes and pathologically accumulates from its normal nuclear locale to the cytosol. Given that the subcellular localization and expression of TDP-43 is tightly regulated, we posit that identifying novel interactors of wild-type and mutant TDP-43 could reveal insight into networks involved in regulating its localization, ultimately driving neurodegeneration in ALS.
Using CRISPR/Cas9, our lab previously generated knock-in cell lines expressing GFP in the endogenous TARDBP locus (encoding for TDP-43) for both wildtype (WT) and an ALS-causing mutant (Q331K). We have shown that the Q331K mutation causes loss-of-function and mislocalization of TDP-43. I performed immunoprecipitation coupled to mass spectrometry (IP-MS) on this cell model to elucidate interactors of WT- and Q331K- TDP-43. Our data show that there is an overall loss of TDP-43 interactors in cells with the TDP-43Q331K mutation. By setting statistical cut-offs for significance, we identified 34 shared and 12 unique interactors of TDP-43WT. We used bioinformatic approaches to identify enriched pathways and literature searches to look for interactors relevant to TDP-43 and ALS pathobiology. Our shortlist of 14 candidates for validation included proteins involved in the nuclear mRNA export pathways, RNA binding proteins and proteins identified in other interactome studies and TDP-43 based screens.
Using orthogonal approaches, we show evidence of robust interaction of four top hits (PABPC1, HNRNPC, DDX39b and ELAVL1) with TDP-43WT, and a significant decrease in the degree of interaction of HNRNPC, DDX39b and ELAVL1 with TDP-43Q331K. Importantly, this decrease in interaction was only observed at the endogenous level, highlighting the importance of maintaining the steady state levels of TDP-43 in the cell for these assays. We characterized the effects of knockdown and overexpression of these four hits using protein-specific overexpression constructs and shRNAs and observed a significant increase in TDP-43 nuclear localization upon knockdown of these four hits, suggesting that there is a functional effect associated with hit knockdown. Overexpression or knockdown of the top hits in a splicing assay did not identify significant changes in TDP-43’s splicing or RNA binding abilities, suggesting that these hits do not affect splicing function in our hit characterization studies.
Using this novel experimental tool and unbiased screen, we identified alterations in TDP-43 protein-protein interactions in the context of ALS and have generated tools to characterize their roles in cellular functions using knockdown and overexpression approaches. Together with the knock-in cells, these tools will allow us to gain insight into pathways involved in driving neurodegeneration, in the context of ALS.
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Tarlarini, C. "MOLECULAR AND GENETIC CHARACTERIZATION OF ALS PATIENTS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232574.
Full textAbstract:
Amyotrophic lateral sclerosis (ALS) is an adult‑onset, rapidly progressive and ultimately fatal
neurodegenerative disorder characterised by degeneration of upper and lower motor neurons.
This leads to weakness, muscular atrophy and spasticity, which relentlessly progress to complete
paralysis with a low survival rate beyond 5 years from symptom onset. In 10% of cases the
disease is considered to be genetically transmitted (FALS) while in the remaining cases it occurs
sporadically in the population (SALS). To date cases of hereditary ALS have been attributed to
mutations in more than 16 different genes, the most common being SOD1, FUS, TARDBP and
C9ORF72; mutations in other genes are rare. The above genes explain 60% of the cases of
familial ALS and 15% of sporadic cases.
The disease can be subdivided into bulbar (25%) and spinal‑onset (75‑80%) forms. Nevertheless,
it is currently recognized that pathological changes are not limited to the motor system: patients
with ALS may exhibit cognitive abnormalities ranging from impaired frontal executive dysfunction to
overt frontotemporal dementia (FTD).
In spite of the above evidence, ALS is regarded as a complex disease in which multiple
environmental and genetic risk factors contribute to disease susceptibility. Furthermore it is
possible that the phenotypic variability, which is frequently detected within families, could be due to
multiple genetic factors as devised in the oligogenic disease model.
In the present study we analysed 285 SALS and 17 FALS cases. Globally, our molecular analysis
explained 10.3% of all ALS cases (31/302). The genes screened were SOD1, TARDBP, FUS and
C9ORF72. Genomic DNA was extracted from peripheral blood through a salting out procedure;
coding regions and exon‑intron boundaries of SOD1 (5 exons), TARDBP (1 exon), and FUS
(5 exons) genes were amplified from genomic DNA and sequenced. Otherwise the repeat‑primed
PCR assay, used for C9ORF72 gene, was performed in order to screen for the presence of the
GGGGCC hexanucleotide repeats expansion in ALS patients. The repeat unit of 6 nucleotides
expands up to more than several hundred times in the affected individuals. Fewer than 10 repeats
are not associated with a pathological phenotype, while more than 30 repeats are associated.
However we still do not know the meaning of intermediate repeat sizes (11‑29). This fact
complicates the attribution of the size expansion to the pathological phenotype observed.
According to the oligogenic disease model, all patients were screened for the most common
ALS‑associated genes and all mutated subjects were tested also for ANG. The affected/unaffected
family members, when available for the study, were screened for SOD1, TARDBP, FUS and
C9ORF72 in order to identify their genetic difference from the proband and to evaluate if this
difference could explain an heterogeneous phenotypic expression of the disease.
Following these analyses we selected and described in detail 5 FALS and 2 SALS cases and
one SETX case, with different intrafamilial phenotypic expression. In one of our SOD1 mutation
carriers, the proband manifested the disease after the delivery; together with a specific angiogenin
genetic variant this condition seems to have anticipated the age of disease onset and contributed
to the aggressive clinical course observed in the proband compared to other family members.
We also found one case in which we observed the phenomenon of anticipation, which could be
due to hormonal treatments together with the simultaneous presence of 2 mutations (C9ORF72/
TARDBP), as suggested in the oligogenic disease model. Indeed, the neuroprotective effects of
estrogens could account for the later age at onset in women as we have tested in another family
harbouring the R521C mutation. In this case a male subject developed the disease, whereas his
older sister didn’t show any neurological signs. In another family we observed a wide spectrum
of clinical symptoms associated with A382T TARDBP mutation. Some family members showed
cognitive impairment without signs of ALS, conversely, other relatives showed a typical ALS without
any signs of dementia. In addition we have evidence of TARDBP mutation carriers without a
neurological phenotype. Surprisingly, two subjects harbouring the mutation in homozygous state
display different spectra of clinical symptoms. The screening of SOD1, FUS, C9ORF72 and ANG
in the family members, to identify other mutations that could explain this intrafamilial phenotypic
heterogeneity, resulted negative; indicating that other genes/conditions could play a role in
ALS disease.
These data support the hypothesis that phenotypic variability seen in patients carrying the same
mutation, could be attributed to additional genetic and/or environmental factors, which could
modify the penetrance of the disease. Furthermore we identified a clinical subgroup of patients
that develop the disease during pregnancy (child-bearing age), thus indicating an additional
modifier conditions linked to hormonal changes. For all these reasons predictive or presymptomatic
testing should be undertaken with caution, especially in unaffected family members. Indeed, it is
impossible for the genetic test to predict clinical outcomes based on the genotypic results only.
A number of uncertainties remain, due to variability in penetrance, expressivity, and modifying
factors, such as gene‑gene interactions and environmental influences. This adds to the debate for
the ethical and psychological dilemmas about genetic testing, since still more has to be discovered
related to this devastating disease.
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Chatzoudis, Susanne, and Anette Wikström. "Personers erfarenheter av att leva med ALS : En beskrivande litteraturstudie." Thesis, Högskolan i Gävle, Avdelningen för hälso- och vårdvetenskap, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:hig:diva-22829.
Full textAbstract:
Bakgrund: Amyotrofisk lateralskleros (ALS) tillhör gruppen motorneuronsjukdomar. Det är en obotlig neurologisk sjukdom som drabbar cirka 200 personer per år i Sverige. De viljestyrda musklerna i kroppen förtvinar vilket leder till att personen får problem med tal och andning. Sjukdomsförloppet varierar beroende på vilken form av motorneuronsjukdom personen drabbas av, vid ALS avlider personen oftast inom 5 år. Syfte: Syftet med litteraturstudien var att beskriva personers erfarenheter av att leva med ALS samt beskriva de inkluderade vetenskapliga artiklarnas undersökningsgrupp. Metod: En beskrivande litteraturstudie som består av 11 kvalitativa artiklar. Huvudresultat: Några patienter var missnöjda med sjukhusvården, de möttes av okunnig personal och blev skickade mellan olika vårdenheter innan diagnosen kunde fastställas. Det ledde till att patienterna själva fick söka efter svar. Patienterna beskrev flera negativa känslor med diagnosen ALS. Under sjukdomsförloppet uppstod även positiva känslor som gjorde att patienterna fortsatte att kämpa. Patienterna använde sig av olika strategier som existentiella tankar, acceptans för hjälpmedel och att ta hjälp av en utomstående vårdare. Stöd från familj och vänner var betydelsefulla för att uthärda sjukdomen och den förväntade döden. Slutsatser: Personer som drabbats av ALS upplevde en känslomässig bergochdalbana, som pendlade mellan positiva och negativa känslor. För flera patienter var det viktigt att ta en dag i taget för att finna meningsfullhet. Sjukdomsupplevelsen är individuell vid ALS och det är viktigt att sjuksköterskan använder sig av personcentrerad vård. Det är betydelsefullt att sjuksköterskan tar till sig patienters och anhörigas erfarenheter för att bevara dess integritet och autonomi.Background: Amyotrophic lateral sclerosis (ALS) belongs to a group of motor neuron diseases. It is an incurable neurological disease that affects approximately 200 people per year in Sweden. The involuntary muscles of the body atrophies which causing the person problems to speak and breath. The disease progression varies depending on the version of motor neuron disease the person suffering, at ALS person usually dies within 5 years. Aim: The purpose of this study was to describe people's experiences of living with ALS and describe the study group of the included scientific articles. Method: A descriptive literature study consisting of 11 qualitative articles. Main Results: Some patients were not satisfied with the hospital care, they were met by ignorant staff and were sent between different care units before a diagnosis could be decided. At the end the patients themselves were searching for answers themselves. Patients describe several negative feelings diagnosed with ALS. During the progression of the disease even positive emotions came and encouraged the patients continue to struggle. Patients used different strategies like existential thoughts, acceptance of aid and help from outside caregiver. Support from family and friends were important to cope with the disease and the expected death. Conclusions: People who suffer from ALS experienced an emotional roller coaster, which commuted between positive and negative feelings. For many patients, it was important to take one day at a time to find meaningfulness. The experience from the illness is individual from ALS and it is important that the nurse uses the person-centered care. It is important that the nurse learn from the patients and relatives experience to preserve the integrity and autonomy.
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Parry, Katherine Elizabeth. "Investigation of the interactions of DVAP-33A, the orthologue of human VAPB." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5538.
Full textAbstract:
Amyotrophic Lateral Sclerosis is the most common type of motor neuron disease, characterized by progressive degeneration of the upper and lower motor neurons. Sufferers present with symptoms of muscle weakness and this quickly develops on to paralysis and finally death due to respiratory failure within 5 years of disease onset. Although the majority of cases are sporadic, about 10% are familial and it is hoped that through the investigation of these few cases a greater understanding of the disease process, the reasons for its delayed onset and vulnerability of motor neurons will be achieved. Recently a novel mutation linked to ALS was discovered in an evolutionary conserved protein named Vesicle associated membrane protein (VAMP) associated protein B (VAPB). VAPB is an integral type II membrane protein localised at the Endoplasmic Reticulum and thought to have a role in protein transport. The orthologue in Drosophila has been shown to be involved in the homeostatic regulation of bouton formation at the Neuromuscular Junction through an association with the microtubule network. To elucidate the mechanism through which this protein causes ALS, Pennetta et al have created a Drosophila model of the disease by expressing the mutated orthologue in the fly. To complement this model, I have undertaken a number of biochemical experiments to look for potential interactors of the VAP proteins. The yeast two hybrid system utilises the yeast GAL4 transcriptional activator to indicate a protein interaction within a yeast cell and can be used to test a cDNA library for interactors. Through this technique a number of interesting binding partners have been found that may play crucial roles in the progression of the disease.