Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder characterised by selective loss of upper and lower motor neurons (MNs). Recently, the GGGGCC (G4C2) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been identified as the most common genetic cause of ALS, highlighting the importance of studying the pathogenic mechanisms underlying this mutation. Accumulating evidence implicates that ALS is a multisystem and multifactor disease. Specifically, non-neuronal cells, astrocytes in particular, are also affected by toxicity mediated by ALS-related mutations, and they can contribute to neurodegeneration, suggesting astrocytes as a key player in ALS pathogenesis. Here, a human induced pluripotent stem cells (iPSCs)-based in vitro model of ALS was established to investigate the impact of the C9orf72 mutation on astrocyte behaviour—both cell- and non-cell autonomous. Work in this study shows that patient iPSC-derived astrocytes recapitulate key pathological features associated with C9orf72-mediated ALS, such as formation of G4C2 repeat RNA foci, production of dipeptide repeat (DPR) proteins and reduced viability under basal conditions compared to controls. Moreover, C9orf72 mutant astrocytes in co-culture result in reduced viability and structural defects of human MNs. Importantly, correction of the G4C2 repeat expansion in mutant astrocytes through targeted gene editing reverses these phenotypes, strongly confirming that the C9orf72 mutation is responsible for the observed findings. Altogether, this iPSC-based in vitro model provides a valuable platform to gain better understandings of ALS pathophysiology and can be used for future exploration of potential therapeutic drugs.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease in which motor neurons selectively degenerate, leading to paralysis and death. Rare causal mutations in FUS and TARDBP implicated RNA binding proteins and RNA metabolism in ALS disease mechanisms. The absence of faithful animal models has impeded precise understanding of the impact of ALS mutations on all functions of ALS-associated proteins. In my graduate studies, I used a novel, animal model of FUS-ALS to explore gain of function disease mechanisms and observed specific, aberrant interactions between mutant FUS and other RNA binding proteins including hnRNP U. Genetic experiments indicate loss of hnRNP U is toxic to motor neurons, suggesting mutant FUS toxicity may result from hnRNP U sequestration and loss of function. In a parallel series of experiments, I also generated novel knock-in mouse models of ALS expressing pathogenic TARDBP mutations to address the flaws of existing model systems and to study the functional consequences of disease-related mutations. We demonstrate that the ALS mutant alleles TDP-43M337V and TDP-43G298S are fully functional and are insufficient to cause age-dependent motor neuron pathology, indicating that physiological levels of mutant TDP-43 are alone insufficient to initiate disease. This model enables future exploration of the interaction between genetic and environmental factors that lead to TDP-43 toxicity in ALS and related disorders. Collectively, our findings suggest a gain of function mechanism of toxicity in which mutations and aging, with other factors, alter the local concentration of RNA binding proteins, leading motor neurons to degenerate.

abstract: In medical imaging, a wide variety of methods are used to interrogate structural and physiological differences between soft tissues. One of the most ubiquitous methods in clinical practice is Magnetic Resonance Imaging (MRI), which has the advantage of limited invasiveness, soft tissue discrimination, and adequate volumetric resolution. A myriad of advanced MRI methods exists to investigate the microstructural, physiologic and metabolic characteristics of tissue. For example, Dynamic Contrast Enhanced (DCE) and Dynamic Susceptibility Contrast (DSC) MRI non-invasively interrogates the dynamic passage of an exogenously administered MRI contrast agent through tissue to quantify local tracer kinetic properties like blood flow, vascular permeability and tissue compartmental volume fractions. Recently, an improved understanding of the biophysical basis of DSC-MRI signals in brain tumors revealed a new approach to derive multiple quantitative biomarkers that identify intrinsic sub-voxel cellular and vascular microstructure that can be used differentiate tumor sub-types. One of these characteristic biomarkers called Transverse Relaxivity at Tracer Equilibrium (TRATE), utilizes a combination of DCE and DSC techniques to compute a steady-state metric which is particularly sensitive to cell size, density, and packing properties. This work seeks to investigate the sensitivity and potential utility of TRATE in a range of disease states including Glioblastomas, Amyotrophic Lateral Sclerosis (ALS), and Duchenne’s Muscular Dystrophy (DMD). The MRC measures of TRATE showed the most promise in mouse models of ALS where TRATE values decreased with disease progression, a finding that correlated with reductions in myofiber size and area, as quantified by immunohistochemistry. In the animal models of cancer and DMD, TRATE results were more inconclusive, due to marked heterogeneity across animals and treatment state. Overall, TRATE seems to be a promising new biomarker but still needs further methodological refinement due to its sensitivity to contrast to noise and further characterization owing to its non-specificity with respect to multiple cellular features (e.g. size, density, heterogeneity) that complicate interpretation. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa, progressiva de início tardio que afeta principalmente os neurônios motores (NM). As causas que levam os NM à morte são variadas e ainda sendo investigadas. A descoberta de alterações genéticas como uma possível causa de ELA deu início à uma nova era na investigação desta afecção. Atualmente existem mais de 30 genes associados com a doença, entre eles o FUS, um gene que frequentemente aparece mutado em casos familiais da doença. A proteína FUS normalmente se localiza predominantemente no núcleo, mas na maioria dos casos de mutações na FUS relacionadas à ELA, ela aparece retida no citoplasma. O presente estudo traz um paciente de ELA (P) portando a mutação p.R521H no gene FUS e três de seus irmãos (dos quais um é portador da mutação e não apresnta sinais clínicos de ELA, e os outros dois não apresentam mutações no FUS) dos quais foram obtidas amostras de sangue e biópsia de pele. O DNA extraído das amostras de sangue, foi submetido ao sequenciamento do tipo Sanger para verificar a presença, ou ausência, da mutação R521H na FUS. A partir dos fibroblastos dos participantes, foram derivadas linhagens de células tronco pluripotentes induzidas (iPSC). As iPSC produzidas passaram por ensaios a fim de indicar o estado de pluripotência e de indiferenciação destas linhagens. Nós investigamos a posição da proteína FUS nas linhagens de iPSC e de fibroblastos e há evidências que, assim como descrito na literatura, a proteína FUS aparece retida no citoplasma das linhagens do paciente e de seu irmão portador da mutação. Desta forma, o presente estudo associa dois irmãos com quadros clínicos discordantes mas que apresentam a mesma mutação e sinais moleculares patológicos semelhantes. As linhagens de iPSC obtidas são um rico material para o uso em pesquisas futuras sobre a ELA / Amyotrophic lateral sclerosis (ALS) is a late onset, progressive, neurodegenerative disease that primarily affects motor neurons (MNs). The causes behind motor neuron death are diverse and still under investigation. The discovery of genetic alterations as possible causes of ALS initiated a new era for ALS research. There are currently over 30 genes associated with the disease, among which is FUS, one of the most frequently mutated in familial cases. The FUS protein is predominantly located in the nucleus, but in most of the ALS-related FUS mutations this protein is dislocated to the cytoplasm. The present work investigates the molecular aspects of a specific FUS mutation, p.R521H. An ALS patient (P) harboring the mutation and three siblings (of which one is a non-affected carrier and two present no mutations in FUS) were analyzed using blood samples and skin biopsies. We extracted DNA from blood samples and submitted it to Sanger sequencing for confirmation of the presence, or absence, of the R521H FUS mutation. The fibroblasts obtained from these biopsies were used for iPSC derivation. Assays were performed to confirm the undifferentiated state and pluripotency for the four strains obtained. We investigated the FUS location in these strains, and there is evidence for FUS retention in the cytoplasm of cells harboring the mutation (as seen in recent literature). Thus, this work associates two siblings with the same pathogenic mutation, showing the same molecular pathological signal but with discording clinical phenotypes. The iPSC strains obtained here are a valuable resource for further ALS investigation

A descoberta de que a expansão da repetição do hexanucleotídeo GGGGCC no gene C9ORF72 é uma das principais causas da Demência Frontotemporal (DFT) e da Esclerose Lateral Amiotrófica (ELA) foi um importante avanço para o entendimento dessas doenças. Essa mutação é responsável por grande parte dos casos hereditários e esporádicos. O indivíduo acometido pode se manifestar clinicamente como ELA, DFT e como a combinação fenotípica ELA-DFT. No entanto, vários outros fenótipos clínicos já foram descritos, como o de doenças que cursam com parkinsonismo atípico, ou mesmo formas que se assemelham à Doença de Parkinson e à Doença de Alzheimer. O objetivo do trabalho foi fazer uma revisão dos fenótipos associados à mutação do gene C9ORF72 descritos na literatura e descrever os casos associados à mutação identificados nos ambulatórios do Hospital das Clínicas de Ribeirão Preto (HCRP). A revisão foi feita a partir de artigos publicados entre 2011 e 2014, buscados na base de dados eletrônica PubMed. Foram selecionados 99 artigos em inglês, em que a mutação do gene C9ORF72 foi objetivo de estudo e, a partir destes, foram selecionados 44 artigos que apresentavam uma descrição individualizada dos fenótipos de um total de 219 pacientes. Nessa revisão, foram identificados 31 fenótipos. No HCRP, os pacientes com sintomas sugestivos eram encaminhados para coleta de sangue após consentimento informado. A extração do DNA a partir do material fornecido pelos pacientes foi realizada no Centro de Medicina Genômica do HCRP de forma automatizada e a amplificação dos fragmentos de interesse foi obtida pela reação de cadeia em polimerase (PCR) e pelo método qualitativo Repeat-Primed PCR (RPPCR). Foram identificados 17 pacientes com a mutação e as manifestações clínicas desses pacientes foram descritas. Foram identificados 6 fenótipos, dentre eles ELA, ELA-DFT, variante comportamental da DFT, Afasia Progressiva associada à ELA, Esquizofrenia e Esclerose Lateral Primária. Conclui-se que a variabilidade da apresentação clínica incial dos indivíduos com a mutação é extensa. Ainda não se sabe o que faz com que a mutação se manifeste de uma forma ou de outra. Saber o real tamanho da expansão do gene que causa essas doenças e ter um maior conhecimento sobre a penetrância do gene são fundamentais para aconselhamento genético das famílias acometidas. / Hexanucleotide repeat expansion GGGGCC in the C9ORF72 gene is one of the main causes of Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) being an important step towards the understanding of these disorders. This mutation is responsible for much of hereditary and sporadic cases. The affected subject may manifest ALS, FTD and ALS-FTD phenotype. However, several other clinical phenotypes have been described as atypical parkinsonism or forms that resemble Parkinson\'s disease and even Alzheimer\'s disease. The objective was to review phenotypes associated with C9ORF72 mutation described in literature and describe cases associated with the mutation identified in outpatient clinics of the Hospital das Clínicas de Ribeirão Preto (HCRP). The review was made from articles published between 2011 and 2014 searched on electronic database PubMed. We selected 99 papers in English in which mutation of the gene C9ORF72 was analyzed, and 44 were selected. We described phenotypes of 219 patients. We found 31 different phenotypes. In HCRP, patients with suggestive symptoms were selected to collect blood after informed consent. DNA extraction from the blood was done by an automated way in Genomic Medical Center in HCRP. Amplification of fragments of interest was obtained by polymerase chain reaction (PCR) and the qualitative method Repeat-primed PCR (RP-PCR). We identified 17 patients with mutation and their clinical manifestations were described. Six phenotypes were described including ALS, ALS-FTD, behavioral variant FTD, progressive aphasia associated with ALS, schizophrenia and Primary Lateral Sclerosis. We conclude variability of initial clinical presentation of patients with mutation is extensive. It is not known why this mutation manifests itself in different ways. Its important to understand how repeat expansion size causes distinct diseases, and to achieve a greater knowledge of the gene penetrance for genetic counseling of affected families.

Motor neuron disease (MND) is a set of neuromuscular diseases that affect the upper and/or lower motor neurons, resulting in progressive disability. Amyotrophic lateral sclerosis (ALS) and Primary lateral sclerosis (PLS) are two forms of MND that both involve upper motor neuron degeneration, which can also accompany extra-motor changes in cognitive, behavioral, and/or emotional functioning for some individuals. Characterization of the cognitive profile of MND is still evolving, with growing interest in cognitive subtypes. The development of cognitive screens targeted to the MND cognitive profile aim to provide efficient and accurate brief assessments. However, empirical evaluation of tailored MND cognitive screens is needed for cross-validation independent of tests’ original developers. The present study addresses the cognitive profile of MND and the utility of brief cognitive screens with a focus on impairments in the language domain. The two primary aims include: (1) comprehensive assessment and characterization of language dysfunction in MND, and (2) empirical evaluation of brief cognitive screens with regard to detecting language impairments. Forty-one patients with MND (ALS n = 36; PLS n = 5) were administered a comprehensive language battery to classify cognitive impairment (MND/ALSci; Strong et al., 2017) in the language domain and/or verbal fluency. Patients also completed two tailored cognitive screens [ALS Cognitive Behavioral Screen (ALS-CBS), Edinburgh Cognitive and Behavioral ALS Screen (ECAS)] and one general screen (Montreal Cognitive Assessment; MoCA). The current preliminary results suggest language dysfunction in MND is characterized by prominent difficulties with word retrieval (confrontation naming) and/or syntax comprehension. However, evidence of reduced word production resembling nonfluent/agrammatic aphasia was not found. In total, 19.5% of the sample met criteria for MND/ALSci in the language domain (n = 8, all ALS); 22.0% met criteria for MND/ALSci in the verbal fluency domain (n = 9). Patients were classified into three subgroups, those with broad language impairments (ALSci-L n = 4, 9.8%), phonemic fluency impairments (MNDci-VF n = 5, 12.2%), or both impairments (ALSci-L+VF n = 4, 9.8%). Results also revealed existing challenges in accurately classifying patients with language dysfunction using brief cognitive screens. The ECAS Language subscore offered limited classification of broad language impairments in the present MND sample (sensitivity 50%, specificity 70%). Among the broader cognitive screens, sensitivities to language impairments were: ALS-CBS (100%), ECAS ALS-Specific Score (75%), and MoCA (71%). Convergent validity was demonstrated between outcomes on the ALS-CBS and ECAS ALS-Specific Score (rФ = .59). Discriminant validity was also demonstrated between outcomes on ALS-CBS compared to the MoCA (rФ = .11). Future research is needed to assess whether language dysfunction reflects a distinct MND cognitive phenotype(s) and potential relationships with disease prognosis. Naming and syntax comprehension may be fruitful language screening targets for future research.

The discovery that the (GGGCC)n>30 repeat expansion in the non-coding region of C9orf72 (C9) is the most prevalent mutation in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has led to a massive effort to discover the mechanism by which the expansion causes ALS. One effect of the repeat expansion is the reduction in C9 mRNA and protein levels. Therefore, we chose to concentrate on studying the function of C9 and how its reduction contributes to ALS progression. First, we show that C9 short (C9S) and long (C9L) isoforms have different cellular localization, and that C9 knockdown in several cell lines, results in significant changes in gene expression. Specifically, we show that differentially expressed genes were enriched in immune system activation pathway. Additionally, we observed gene expression changes in important glutamate-glutamine cycling genes and show that C9 knockdown results in accumulation of intracellular glutamate. We also show that C9S isoform may regulate gene expression as it interacts with chromatin, and can be ChIPed on the promoter of endothelin-1 (EDN1). C9 knockdown also leads to significant morphological changes that include increased cell sizes and nucleus, massive vacuolization, and results in reduced cell viability. Investigation into the vacuoles revealed that they originate from hyperactivation of macropinocytosis. The hyperactivation of macropinocytosis results in a caspase-independent cell death known as methuosis. We show that vacuolization is a p53-dependent process, and we present evidence that p53 mediates vacuolization via the repression of the mevalonate pathway. Furthermore, we found that inhibition of isoprenylation, a process depending on mevalonate pathway products (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), participates in the induction of vacuoles. Importantly, we also reveal that C9 knockdown leads to mitochondrial dysfunction, increased ROS, increased DNA-damage and p53 activation, all of which are seen in C9 ALS patient samples or in C9 patient derived motor neurons (C9 iMNs). Our results reveal several previously unknown pathways which are affected by C9 knockdown , which have potential therapeutic implications, that include endothelin signaling and macropinocytosis, both of which can be blocked with FDA approved drugs.

Doctor of Philosophy / Abstract Neurodegenerative disorders are a diverse group of disorders that affect specific subsets of neurons. Motor neuron diseases, neurodegenerative disorders of motor neurons, are seen commonly as sporadic cases and less frequently as familial disease forms. The familial forms show genetic and phenotypic heterogeneity. Clinically motor neuron diseases may be seen as rapidly progressive disorders like amyotrophic lateral sclerosis, ALS or slowly progressive disorders like hereditary motor neuropathies, HMN. The only proven causes for motor neuron diseases are gene mutations that lead to motor neuron degeneration in familial disease forms. Only some of these genes have been identified and have contributed greatly to our understanding of the neurobiology of familial and sporadic disease forms. Identification of additional disease causing genes would help enhance our knowledge of the pathophysiological mechanisms underlying all forms of motor neuron disorders, which would lead to early diagnoses, effective prophylaxis and efficient therapies for these disorders. This study aimed to find gene mutations that cause rapid and slowly progressive familial motor neuron disorders in Australian families and to determine their relevance to sporadic forms of motor neuron disease. The familial forms of ALS show reduced disease penetrance, that is, not all gene mutation carriers manifest the disease. This study examines ALS penetrance in a group of Australian families. The most frequently observed mutations in ALS families are cytosolic superoxide dismutase/SOD1 gene mutations. In a collection of ALS families in our centre, families without the common SOD1 gene mutations were genotyped for other ALS genes and loci and studied using genetic linkage and haplotype analyses. Studies in a large Australian ALS family further confirmed genetic heterogeneity in non-SOD familial ALS, all known autosomal dominant ALS genes and chromosomal loci were excluded as cause of disease in this family. Such families can be studied further to identify additional disease genes and loci mapped in other ALS families. These families represent powerful resources for identification of additional ALS genes. Identifying the pathogenic genes in families with reduced disease penetrance may be more relevant to sporadic forms of disease. dHMN is a chronic neurodegenerative disorder predominantly affecting motor neurons. In a large Australian dHMN family, all the known dHMN genes and chromosomal loci were excluded as cause of disease. A genome wide microsatellite screen was performed in this family and genetic linkage was established to a novel 12.98 Mb locus on chromosome 7q34.2-q36. Candidate genes in this large interval will be screened based on their function and expression profile. Identification of a new dHMN locus provides the basis for future identification of a novel gene involved in motor neuron degeneration. Genes in dHMN have been shown to be pathogenic in ALS and Charcot Marie Tooth syndromes. The new locus for dHMN mapped in this project would lead to identification of a novel dHMN gene, which may elucidate the pathogenesis underlying a wide range of neurodegenerative disorders.

<p>Syftet med denna studie var att beskriva hur människor med Amyotrofisk lateralskleros (ALS) upplever sin livskvalitet. Metoden som användes var en litteraturstudie med beskrivande design. Olika kombinationer av sökorden ”ALS”, ”amyotrophic lateral sclerosis” ”quality of life”, och ”människor” användes vid sökning i databaserna Medline (PubMed) och Science Direct. Totalt analyserades tretton vetenskapliga artiklar som sedan kvalitetsbedömdes och kategoriserades i fyra kategorier: fysisk, psykisk, social och existentiell livskvalitet. Forskning kring de fysiska aspekterna av livskvalitet visade att det främst är den nedsättande funktionsförmågan och begränsningarna relaterade till sjukdomen som bidrar till det fysiska lidandet. Med tiden ändrades patienternas intressen från mer aktiva till passiva. Till skillnad från detta var upplevelsen av det psykiska lidandet mycket låg. Resultat tillhörande den sociala aspekten visade att patienternas förhållande med familj och vänner var av störst värde för att uppleva livskvalitet samt det främsta stödet för att bemästra de svårigheter som sjukdomen medför. Patienternas religiösa tro och tillgång till präst var av betydande roll vad gällande bemästringen av existentiella frågor och funderingar.</p> / <p>The purpose of this study was to describe how people with Amyotrophic lateral sclerosis (ALS) experience their quality of life. The method used was a study of literature with design description. A various combination of search words, such as “amyotrophic lateral sclerosis”, “ALS”, “quality of life” and “people” were used in searching the computer bases Medline (Pubmed) and Science Direct. In total, thirteen scientific articles were analysed and quality tested, subsequently they were categorized into four categories: physical, mental, social and existential quality of life. Research into the physical aspects surrounding the quality of life showed that it is, above all, the disparaging functional capabilities and restrictions related to the illness which causes the mental suffering. In time, the patients interests changed from active to passive. In contrast to this, the experience of mental suffering was very low. Results of the studies into the social aspects showed that the patients relationship with family and friends was of the greatest value to quality of life, as well as the foremost support towards conquering the difficulties which the illness causes. The patients religious beliefs and access to a priest, play a significant part regarding the conquering of existential questions and thoughts.</p>

<p>Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation.</p><p>An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease.</p><p>The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.</p>