The fused in sarcoma (FUS) protein has been shown to be a significant disease protein in a subgroup of patients with frontotemporal lobar degeneration (FTLD). Nevertheless, the mechanism underlying FUS associated FTLD is only poorly understood. Recent research has identified a large hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72), reinforcing the association between C9orf72 and FTLD. Moreover, an unusual histopathological change has been observed within the granule cell layer of the cerebellum in chromosome 9p-linked frontotemporal dementia with motor neuron disease. Whether this type of cerebellar pathology is a pathological marker for chromosome 9p-linked families remains unknown. The purpose of this study was to genetically, neuropathologically and biochemically characterize FUS and C9orf72 in FTLD, and also to investigate the association between the cerebellar pathology and chromosome 9p-linked families. The genetic sequencing study searching for potential genetic factors of FUS in FTLD failed to detect any pathogenic mutations or variations. Immunohistochemical study for FUS pathology in FTLD provided strong evidence for FUS being the specific pathological protein in all forms of FTLD-FUS. Immunoblotting for FUS in FTLD detected one novel disease-associated FUS aggregate (~37 kDa) in the urea fraction of atypical FTLD with ubiquitinated inclusions (aFTLD-U) frontal cortical samples, suggesting this unique protein product might be more associated with disease than the full-length protein itself. Immunohistochemical study of C9orf72 in FTLD detected a 'synaptic' staining in CA sectors, as the most prominent histological feature identified. Immunoblotting for C9orf72 protein demonstrated no distinctive bands among different diagnostic groups, in frontal and cerebellar cortical regions. The present study also confirmed the presence of cerebellar p62 neuronal cytoplasmic inclusions (NCI) in a proportion of FTLD-TDP cases. Although most of these cases showed an autosomally dominant pattern of inheritance, not all of them shared a common C9orf72 haplotype, or mutation in C9orf72.Much work is still needed to investigate the underlying pathogenesis of FTLD-FUS. Attention should still be given to identifying possible genetic risk factors in FUS using a large series of FTLD samples and searching for other possible proteins within the FUS immunoreactive neuronal inclusions. Moreover, the target protein within the cerebellar p62 NCI remains unknown, but it is clear that it is not C9orf72 protein.

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.

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.

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.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that affects corticospinal and spinal motor neurons and leads to death within 30 months of symptom onset in half of all cases. It remains incurable and treatment is supportive. The genetic and molecular understanding of ALS has gone through a rapid expansion in recent years, notably with the discoveries of TDP-43, a heterogeneous ribonucleoprotein as a major component of neuronal inclusions in ALS, as well as the discovery of the C9orf72 hexanucleotide expansion as the most common genetic cause of this disease. This first part of this thesis addresses the question of which of the various pathological hallmarks of the C9orf72 Hexanucleotide Repeat Expansion (HRE) in autopsy material correlates best with the clinical presentation. The main finding is that TDP-43 distribution, rather than C9orf72 RNA foci or dipeptide aggregation in the brain, corresponds best with the areas relevant to the clinical subtype of ALS-FTD. Subsequently the role of TDP-43 was investigated in induced pluripotent stem cell derived motor neurons, and no evidence of the hallmarks of TDP-43 dysfunction, were seen in this model of the disease. No mislocalisation is found on immunofluorescence, and biochemical analysis shows no differences in insoluble species between the patient and control cell lines. In the final section, RNA sequencing was used to study the transcriptome of a BAC transgenic mouse carrying a human M337V transgene expressed at low levels, to identify early presymptomatic differences in gene expression. Interestingly, no changes were found in genes known to be associated with ALS through mutations, and the constitutive nuclear functions of TDP-43 in the regulation of splicing was maintained, prior to the emergence of a clinical phenotype in the mouse. This favours a gain of function mechanism for TDP-43 mutations in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Microsatellite repeats are a phenomenon found in DNA where a short sequence, usually 1-6bps, is repeated dozens to hundreds of times. Microsatellite repeats that are able to be transcribed are termed expanded tandem repeat-containing RNA (xtrRNA) [1]. xtrRNA have been associated with many diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), which are both caused by a repeat in the C9ORF72 gene. Recent research has been focused on trying to provide treatments for patients with these diseases. This study focuses on creating a drug screening process for therapeutics targeting transcription by stopping or slowing the transcription of C9ORF72 repeat expansions. One project has focused on interrupting the interaction of two transcription factors, SUPT5H and SUPT4H1, to slow transcription. Another project has focused on slowing transcription by using transcriptional inhibitors or nucleoside analogs at low concentrations. Our hypothesis is that if transcription rates are slowed enough, pausing or arrest of RNA polymerase will be induced at complex sequences, including GC-rich regions and repeats. This should reduce synthesis of xtrRNA and provide a starting point for therapeutic development.

The hexanucleotide repeat expansion GGGGCCn (also known as G4C2n) localizes in the first intron of the C9ORF72 gene and is the most common genetic cause of ALS and FTD (C9ALS/FTD). The pathomechanisms proposed for C9ALS/FTD suggest that from sense (G4C2)n- and anti-sense (C4G2)n-containing transcripts originate two different mechanisms of toxicity: i) by the alteration of RNA processing due to binding and sequestration of RNA-binding proteins, thereby leading to impairment of RNA metabolism; and ii) by their unconventional Repeat-associated non AUG (RAN) translation into five different dipeptide-repeats (DPRs). In addition, pathological expansion of (G4C2)n reduces the C9orf72 transcription causing loss of function of the C9ORF72 protein. The toxicity of some of these DPRs has been showed in several cell lines, in iPSC-derived neurons, in Drosophila and in mouse models. An impairment of the ubiquitin-proteasome system (UPS) due to aggregation of toxic proteins is largely demonstrated in neurodegenerative disorders and among the mechanisms of DPR-related toxicity. RAN translation of (G4C2)n-RNAs has been recently shown to require a near-cognate start codon upstream of the repeat in frame +1 and to be triggered by stress conditions in a cap-dependent or cap-independent way. However, the mechanism regulating RAN translation is still largely unknown. Importantly, no small molecules are known to selectively modulate RAN translation, even if antisense oligonucleotides (ASOs) and small molecules binding the r(GGGGCC)n have been proposed as therapeutics for C9ALS/FTD. In addition, no effective pharmacological approach to reduce the pathological load of DPRs is currently available. Here, I developed a high-throughput drug-screening assay to identify small molecules and relative molecular targets that can modulate the DPR level. Among the identified hits, two hits reduced DPRs expression levels triggering the protein clearance system in vitro. Moreover, the screening identified compounds having the same target that increased DPRs expression levels indicating the targeted pathway as a crucial modulator of the translation process of the C9orf72 repeat-containing mRNAs. Conversely, I showed that pharmacological inhibition of the pathway reduced DPRs expression levels in vitro, while in vivo it rescued climbing ability and increased life span of Drosophila flies carrying G4C2X36 repeats. Moreover, genetic ablation of the target reduced DPRs expression levels by decreasing their translation efficiency in vitro and rescued the pathological phenotype in vivo. Together, the results show the identification of new pathways as new drug targets for C9ALS/FTD.

More than 40 neurological diseases are known to be caused by large expansions oftandem repeat sequences scattered throughout the human genome in introns, exons and untranslated regions. The GGGGCC (G4C2) repeat expansion located in the first intron of the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In C9 FTD/ALS, expanded transcripts are known to aggregate and accumulate in the cell nucleus, sequestering RNA binding proteins. Other expanded RNA species are exported to the cytoplasm to undergo a non-canonical form of translation termed ‘repeat-associated non-AUG (RAN) translation’. RAN translation leads to the production of toxic polydipeptide repeat proteins in the absence of a canonical AUG start codon. This dissertation will highlight new mechanistic features of translation across the G4C2 repeat expansion, identify a potential therapeutic for C9 FTD/ALS using RNAi and develop a cellular system to explore the G4C2 repeat RNA lifecycle. First, we demonstrate that increasing G4C2 repeat expansion size results in suppression of translation from both canonical and non-canonical start codons, suggesting that large polydipeptide repeats are rarely fully translated. We further find that initiation does not occur from within the repeat expansion, relying on upstream sequence for initiation. However, some reading frames are prone to substantial frameshifting, such as poly-GA. We also show that a bias in ii codon usage efficiency contributes to previously observed variations in the levels of each polydipeptide. Our results support and extend previous studies by identifying two new mechanisms that bias production of poly-dipeptides toward poly-GA in C9 FTD/ALS. Further, we generated central mismatch-containing short hairpin RNAs (shRNAs) targeting the G4C2 repeat expansion to reduce aggregation or block translation of repeatcontaining transcripts. Iterative design was able to improve shRNA processing efficiency and cellular abundance, yet they were unable to reduce nuclear RNA foci in patient-derived cells. Despite this, we show preliminary data suggesting that these shRNAs are able to target cytoplasmic repeat-containing transcripts and resulting in a reduced translation of poly-GP. Finally, we optimized the previously published RNA-protein interaction detection (RaPID) technique, which uses proximity dependent labelling by a mutant biotin ligase and mass spectrometry for protein identification in living cells, to identify proteins interacting with the G4C2 repeat expansion. We embedded the box B RNA hairpin between G4C2 repeats and tested the ability for λN fused to a biotin ligase mutant, BASU, to specifically bind the box B hairpin in vitro. We show that 6 repeats each side of the hairpin combined with an extended hairpin stem promotes specific binding of the λN-BASU fusion protein and is likely to be successful in cells. C9 FTD/ALS is a currently incurable neurodegenerative disorder largely due to the limited understanding of disease mechanism. This dissertation demonstrates new mechanisms of translation across the G4C2 repeat expansion that results in toxic DPR production while also developing a nucleic acid therapeutic for long-term treatment of C9 FTD/ALS and further developing systems to explore RNA-mediated toxicity in cells.

Les dégénérescences lobaires frontotemporales (DLFT) représentent la deuxième étiologie neurodégénérative chez l’adulte de moins de 65 ans. Les DLFT sont constituées d’un ensemble hétérogène de phénotypes cliniques et sont fréquemment héréditaires. Leurs particularités neuropathologiques communes reposent sur une atrophie des lobes frontaux et/ou temporaux associée à la présence d’inclusions de protéines agrégées parmi lesquelles la protéine TAR DNA binding protein 43 (TDP43). Actuellement, aucun marqueur protéique n’est validé pour diagnostiquer les DLFT du vivant du patient.Une cohorte de cas certains DLFT-TDP43 a été constituée grâce au développement d’outils spécifiques de diagnostic moléculaire. Une analyse des concentrations pondérales de protéine TDP43 dans le liquide cérébrospinal (LCS) a été réalisée dans cette cohorte, puis comparée à des cohortes bien caractérisées sur le plan clinique et neuropathologique. Finalement, les profils qualitatifs de la protéine TDP43 ont été étudiés dans différents compartiments accessibles du vivant du patient : les profils des formes solubles (LCS et plasma) et des formes intracellulaires (éléments figurés du sang) de la protéine TDP43 ont été comparés aux profils protéiques obtenus sur des tissus cérébraux présentant des inclusions de protéine TDP43. Les profils protéiques des culots plaquettaires présentent des similitudes avec le tissu cérébral et pourraient devenir un marqueur candidat pour le diagnostic probabiliste des DLFT / Frontotemporal lobar degeneration (FTLD) syndrome is the second most common of presenile dementia. FTLD is a clinically heterogeneous syndrome and comprises many hereditary cases. Common neuropathological features rely on a degeneration of the frontal and/or anterior temporal lobes, associated to specific inclusions of aggregated proteins including TAR DNA binding protein 43 (TDP43). Unfortunately, no practical protein marker is currently validated to improve FTLD diagnosis in living patients.A cohort of FTLD patients with definite TDP43 pathology was defined with the development of specific genetic testing. An analysis of TDP43 concentrations in cerebrospinal fluid (CSF) was performed in this cohort and then compared to other cohorts well-characterized on clinical and neuropathological features. Finally, qualitative patterns of TDP43 were studied in compartments accessible from the patient’s living: profiles of soluble TDP43 protein (in CSF or in plasma) and intracellular TDP43 protein (in the formed elements of blood) were compared to protein patterns observed in brain tissues with TDP43 protein inclusions. Platelet samples exhibit similar characteristics to brain tissue and could become a candidate biomarker for FTLD probabilistic diagnosis