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Genetic Analysis of Familial Alzheimer's Disease, Primary Lateral

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Genetic Analysis of Familial Alzheimer's Disease, Primary Lateral Genetic analysis of familial Alzheimer’s disease, primary lateral sclerosis and paroxysmal kinesigenic dyskinesia: a tool to uncover common mechanistic points Author Jacek Szymański Doctoral programme in neurosciences Director Tutor Jordi Pérez-Tur Alicia Salvador Fernández-Montejo November 2020 Dr Jordi Pérez-Tur, Investigador Científico del Instituto de Biomedicina de Valencia (IBV- CSIC), en calidad de Director de la tesis doctoral de Jacek Szymański, adscrito al Programa de Doctorado en Neurociencias de la Universitat de València. CERTIFICA Que la tesis titulada “Genetic analysis of familial Alzheimer’s disease, primary lateral sclerosis and paroxysmal kinesigenic dyskinesia: a tool to uncover common mechanistic points” se ha desarrollado bajo su dirección y supervisión, y que el trabajo de investigación realizado y la memoria del mismo, ha sido elaborada por el doctorado y cumple los requisitos científicos y formales para proceder al acto de defensa de la Tesis Doctoral. Y para que conste, en el cumplimiento de la legislación presente, firman el presente certificado en València, 2020. ___________________ ____________________ ____________________ Dr Jordi Pérez-Tur Dra Alicia Salvador Jacek Szymański Director Fernández-Montejo Doctorando Tutor Académico ACKNOWLEDGEMENTS This thesis is dedicated to Hanna Szymańska, the person to whom I owe my decision to pursue a doctoral degree and whose knowledge of science and fullest support I could always count on. I would like to express my deepest appreciation and gratitude to my supervisor and director of this thesis, Dr Jordi Pérez-Tur, for his valuable advice, unparalleled support, constructive criticism and guidance. He accepted me under his wing, created a wonderful work environment and is undoubtedly the best group leader I have encountered in my scientific career. I am deeply indebted to Fernando Cardona, without whose support and nurturing, this thesis would not have been possible. Always ready with helpful advice and practical suggestions, I am extremely grateful to him, and I am fortunate to have met him. I am thankful to CIBERNED (Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas) for funding this research into such important neurodegenerative diseases. I am also grateful to all my colleges at IBV-CSIC (Instituto de Biomedicina de Valencia - Consejo Superior de Investigaciones Científicas) who supported me with great advice and camaraderie through the years. Especially Jessica Valdivia, Conchi Rubio and Guillermo Casero, who helped me with my work, brightening up my day with great humour. I would like to thank Małgorzata Wiweger and Jacek Kuźnicki who accepted me for a training stay at the IIMCB (International Institute of Molecular and Cell Biology) in Warsaw, which greately improved my knowledge and abilities. I would like to acknowledge the assistance and collaboration of Ana Espinosa and Teresa Caballero Vizcaíno from the IBMCP-CSIC (Institute for Plant Molecular and Cell Biology), and Jerónimo Bravo and Manuel Soriano from IBV-CSIC. Also the contribution of clinicians: Juan Francisco Vázquez Costa and Juan A. Burguera from Servicio de Neurología, Hospital Universitario y Politécnico de La Fe, and Àngel Pérez Sempere from Hospital General Universitario de Alicante. Special thanks to my family, Maciej, Hanna, Katarzyna, Michał, Tomasz, Keri, Dickson, Mały and Karo for unwavering support and endless good cheer and generosity. In particular, I want to thank Agnieszka, whose support cannot be overestimated, who kept me motivated, patient, and most of all happy. Finally, I am grateful to my friends, like Luke, for a healthy work–life balance. Wiara w daktyla nie jest daktylem samym w sobie – Hanna Szymańska SUMMARY Introduction Neurological diseases can have a wide spectrum of phenotypes, causing disruptions in daily life activities and, often, a progressive disease ending in loss of life. Molecular mechanisms underlying many of these diseases can have common pathways and depending on the gene in question, its dysfunction may lead to a variety of conditions. Alzheimer's disease (AD) is the most common neurodegenerative dementia in the World with an estimate of 50 million people living with dementia worldwide. Symptoms of AD include difficulty remembering names or recent events, apathy, depression, disorientation, confusion, difficulty speaking, walking and swallowing. The canonical division of AD into early onset (EOAD) and late onset (LOAD) is set at 65 years of age. Both are characterised by intracellular hyperphosphorylated tau protein aggregates called neurofibrillary tangles (NFTs) and extracellular senile plaques composed primarily of clumps of amyloid-β (Aβ) peptide. The AD pathology starts at the entorhinal cortex, a region of the medial temporal lobe and causes neuron death leading to atrophy of brain tissue. AD is a multifactorial disease with a dichotomous pattern of inheritance with approximately 70 % of the causes being genetic and the rest environmental. Mutations in genes: APP, PSEN1 and PSEN2, are the most common causes of EOAD and allele ε4 of APOE is a well-established risk factor of LOAD. Three main pathways encapsulate most of the AD genetic risk factors: the immune response, lipid metabolism and endocytosis. Among the genes involved in immune response, CR1 has been associated with AD through genome-wide association studies (GWAS) and is an important part of the complement system, and significant for this work. A particular CR1 isoform, CR1*2, is expressed at lower levels than isoform CR1*1 and it is speculated it affects lower Aβ clearance and dysregulation of the complement system. Primary lateral sclerosis (PLS) is considered as part of the amyotrophic lateral sclerosis (ALS) pathological spectrum. ALS, although considered a rare disease, is the most common motor neuron disease (MND). PLS is characterised by spinobulbar spasticity caused by upper motor neurons (UMNs) degeneration, while ALS is characterized by degeneration of both UMNs and lower motor neurons (LMNs) at spinal and bulbar level, causing limb paralysis, dysarthria, dysphagia and fatal respiratory failure. Clinical features of PLS include spasticity, slight weakness in the lower limbs, adult-onset, progressive course, duration of longer than 4 years and pseudobulbar symptoms. Behavioural and cognitive deficits may occur with ALS, ranging from mild, moderate to frontotemporal dementia (FTD) which was also seen in patients with PLS. The genetic basis of PLS is not well understood although mutations in genes SPG7 and TBK1 were reported in patients affected by familial PLS. Mutations in SOD1 gene, encoding an antioxidant enzyme, and in the protein encoded by C9orf72 gene are the most common causes of ALS. ALS pathogenesis may be caused by glutamate excitotoxicity, mitochondrial dysfunction, impaired structure or transport in axons and oxidative stress. Paroxysmal kinesigenic dyskinesia (PKD), the most common paroxysmal movement disorder, is characterized by a range of involuntary movements triggered by sudden motion. With onset in childhood or adolescence its clinical features include recurrent attacks involving chorea, athetosis, dystonic postures or ballismus. Severity of these attacks typically decreases with age. Mutations in gene PRRT2 are thus far the only cause of this disorder. PRRT2 is known to interact with proteins SNAP-25, SYT1 and SYT2 in the presynaptic membrane of neurons, which are involved in signalling in nerve cells. The lack of PRRT2 often caused through nonsense-mediated mRNA decay (NMD) pathway, due to premature termination codons (PTCs) in the transcript, is said to be the common molecular mechanism involved in haploinsufficiency causing PKD. Objectives The main objective of this work is to comprehend the etiopathogenesis of three neurological diseases affecting three distinct Spanish families and to study the genetic and molecular mechanisms affecting these diseases. Materials and methods DNA samples were collected from members of three Spanish families: UGM037 (11 individuals) affected by AD, UGM471 (7 individuals) affected by PLS and UGM478 (7 individuals) affected by PKD. A control population of healthy individuals and individuals with AD was also available and all the samples were collected with approval of the corresponding institutional review boards of the corresponding hospitals with signed informed consent from patients. The bacterial strain used for all necessary manipulations was Escherichia coli DH5α and the human cell line was SH-SY5Y. Whole-exome sequencing (WES) was used as the tool to identify variants within the exome of the patients for further investigation. Through base-calling and image analysis, read alignment and SNP calling the data could be transformed into a workable database of variants. Filtering and prioritization followed by Sanger sequencing validation of the results was used to identify the most interesting variants, possibly involved in causing the disease. Public databases such as Collaborative Spanish Variant Server (CSVS) or Genome Aggregation Database (gnomAD) were used for variant assessment. The variant frequency was also verified through allele-specific PCR (ASPCR) performed on the control populations. After identifying the most interesting variants, plasmid manipulations were performed to obtain specific cDNAs, corresponding to sequences of the genes with these variants, in specific expression vectors. Site-directed mutagenesis was used for introduction of single nucleotide variants (SNV), FLAG
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