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.

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 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.

Neurodegenerative disorders are a group of age-related conditions resulting in neuronal cell death and protein accumulation. It is estimated that around 5-10% of these cases are genetically mediated. Most commonly this is by pathogenic single nuclear variants (SNVs), though combinations of rare variants (termed oligogenic variation), copy-number variation (CNVs), somatic mutations in nuclear DNA, and somatically acquired mitochondrial DNA variants have all been hypothesised to increase disease risk or cause disease. Firstly, using a combination of exome sequencing and array genotyping on 1511 post-mortem brain samples within the MRC Brain Bank, we detected 61 monogenic cases of disease, 349 brains carrying disease risk factors, and identified that variants in GRN and PRPH may increase the risk of developing dementia with lewy bodies (DLB) and Alzheimer’s disease (AD) respectively. Secondly, we detected a previously unknown systematic bias in the interpretation of oligogenic interactions with implications for our understanding of disease mechanisms and coexistent clinical diagnostic utility. Thirdly, we detected a novel copy-number gain in LAMA5 associated with Creutzfeldt-Jakob disease (CJD), and fourthly, we determine that at least 1% of the population carry high level somatic protein-coding mutations affecting at least 10% of cells within the brain. Subsequently, additional focussed deep-sequencing studies revealed that several regions of the brain are likely to contain clones of low-level somatic mutations that are pathogenic when present in the germline, and that age-related clonal mutations that arise in blood are present at high levels within the aging brain and are associated with Lewy Body pathology. Finally, using transgenic mice that over express human α-synuclein and which either accrue or transmit mtDNA mutations, we determine that the presence of mtDNA mutations exacerbate some phenotypic traits of Lewy body disorders, and may reduce the volume of critical neuroanatomical brain regions whilst paradoxically reducing α-synuclein accumulation. Taken together, these data enable the first genetically stratified brain tissue resource in the UK, describe new disease genetic risk factors (both SNVs and CNVs) for neurodegenerative disorders, and also help define the somatic genetic architecture of the human brain. In addition, we describe the in vivo interaction between mutations in the mitochondrial genome and a progressive neurodegenerative disorder in mice.

A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.

Orientadora: Roberta Gonçalves da Silva / Banca: Luciana Pinato / Banca: Kátia Flores Genaro / Resumo: A disfagia orofaríngea é sintoma frequente em indivíduos com Doença do Neurônio Motor (DNM) e um dos parâmetros que mensuram o grau de funcionalidade nessa população, sendo que a alteração na fase oral da deglutição é um dos primeiros sinais da disfagia na DNM. O comprometimento na fase oral da deglutição é ocasionado por fraqueza, fasciculação e atrofia de língua que promovem incoordenação oral gerando aumento no tempo de transito oral (TTO), comprometendo assim a alimentação destes indivíduos. Objetivo: Esta pesquisa teve por objetivo descrever e relacionar o TTO da deglutição com o desempenho funcional e com penetração e aspiração laringotraqueal na DNM. Método: Participaram desse estudo 20 indivíduos com DNM, independente do tipo ou tempo da doença. Foram incluídos 11 indivíduos, sendo nove do gênero masculino e dois do gênero feminino, faixa etária de 31 a 87 anos (média de idade de 57 anos). Foram excluídos nove indivíduos. Aplicado o questionário Amyotrophic Lateral Sclerosis Functional Rating Scale - revised (ALSFRS-r/BR) para classificação dos indivíduos de acordo com parâmetros Global, Bulbar e Bulbar/Respiratório. Realizada videofluoroscopia da deglutição com diferentes consistências de alimento no volume de cinco ml, com análise quantitativa temporal por meio de software específico para mensuração do tempo de trânsito oral total (TTOT) e análise qualitativa por meio da escala de Penetração e Aspiração Laringotraqueal proposta por Rosenbek et al (1996). Para relac... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Oropharyngeal dysphagia is a frequent symptom in individuals with motor neuron disease (MND) and one of the parameters that measure the degree of functionality in this population, and the alteration in the oral phase of swallowing is one of the first signs of dysphagia in DNM. The impairment in the oral phase of swallowing is caused by weakness, fasciculation and tongue atrophy that promote oral incoordination leading to an increase in oral transit time (OTT), thus compromising the feeding of these individuals. Objective: This study aimed to describe and correlate oral transit time (OTT) of swallowing with functional performance and with laryngotracheal penetration and aspiration in MDN. Method: Participated in this study 20 individuals with DNM, regardless of the type or time of the disease. Eleven individuals were included, nine of whom were male and two were female, ranging from 31 to 87 years old ( mean age of 57 years). Were excluded nine individuals. Applied the Amyotrophic Lateral Sclerosis Functional Rating Scale - revised (ALSFRS-r / BR) to classification of individuals according to Global, Bulbar and Bulbar / Respiratory parameters. A videofluoroscopy of swallowing with different food consistencies was performed in the volume of five ml, with qualitative analysis using the Laryngotracheal Penetration and Aspiration Scale proposed by Rosenbek et al (1996) and temporal quantitative analysis using specific software to measure total oral transit time (TOTT... (Complete abstract click electronic access below) / Mestre

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23218号 / 工博第4862号 / 新制||工||1759(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 庸裕, 教授 跡見 晴幸, 准教授 菅瀬 謙治, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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.

The retromer is a protein complex whose function is to mediate the recycling of proteins from the endosome to either the plasma membrane or the trans-Golgi network. A deficit in retromer function has been associated with multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). In both AD and PD, deficiencies have been found in retromer expression both in patient tissues and in animal models of disease. Furthermore, mutations in the retromer and in retromer-associated genes have been strongly linked with both diseases. Despite ample evidence of the link between the retromer and neurodegeneration, little is known about the retromer in the context of amyotrophic lateral sclerosis (ALS), another common neurodegenerative disorder. ALS is an adult-onset neurodegenerative disorder of the upper and lower motor neurons (MNs) characterized by muscle wasting and weakness leading to death within 3-5 years after diagnosis. To date, the most commonly used model of ALS is a transgenic (Tg) mouse that overexpresses an ALS-causing G93A mutation in the human superoxide dismutase 1 (SOD1) gene. In this study, I first establish a link between the retromer and ALS by showing that cells from ALS patients as well as tissues and cells from SOD1G93A-Tg mice express lower protein levels of the retromer core components—vacuolar protein sorting 35 (Vps35), Vps26a, and Vps29. I then establish that deficiencies in retromer core proteins have functional consequences in an in vitro model of ALS. Having found significant deficiencies in the retromer in SOD1G93A-Tg mice, I then followed the model of studies performed in mouse models of other neurodegenerative disorders by investigating whether repletion of retromer levels, either virally or pharmacologically, in SOD1G93A-Tg mice confers a therapeutic benefit. Surprisingly, I find that rather than ameliorating disease, repletion of retromer levels in SOD1G93A-Tg mice exacerbates it, resulting in a faster decline in motor performance, earlier mortality, and a decrease in MNs in the spinal cord. Finally, since retromer repletion causes deleterious effects on SOD1G93A-Tg mouse disease progression, I study the effect of a single allele deletion of Vps35 in SOD1G93A-Tg mice and find that this depletion of the retromer results in amelioration of disease, including delayed onset of symptomatology, slower decline of motor deficits, delayed mortality, and an increase in MNs in the spinal cord. Altogether, the findings reported herein, support the notion that a mild defect in retromer develops over the course of the disease, which, rather than being deleterious may be therapeutic in mutant SOD1-induced MN degeneration. Perhaps this unexpected outcome may be explained by the fact that the observed mild nature of the defect is not sufficient to kill MNs but enough to alter the trafficking of specific cargos such as AMPA receptors, allowing MNs to better withstand the neurodegenerative process.

Objective. The P300 speller is a brain-computer interface (BCI) that can possibly restore communication abilities to individuals with severe neuromuscular disabilities, such as amyotrophic lateral sclerosis (ALS), by exploiting elicited brain signals in electroencephalography (EEG) data. However, accurate spelling with BCIs is slow due to the need to average data over multiple trials to increase the signal-to-noise ratio (SNR) of the elicited brain signals. Probabilistic approaches to dynamically control data collection have shown improved performance in non-disabled populations; however, validation of these approaches in a target BCI user population has not occurred. Approach. We have developed a data-driven algorithm for the P300 speller based on Bayesian inference that improves spelling time by adaptively selecting the number of trials based on the acute SNR of a user's EEG data. We further enhanced the algorithm by incorporating information about the user's language. In this current study, we test and validate the algorithms online in a target BCI user population, by comparing the performance of the dynamic stopping (DS) (or early stopping) algorithms against the current state-of-the-art method, static data collection, where the amount of data collected is fixed prior to online operation. Main results. Results from online testing of the DS algorithms in participants with ALS demonstrate a significant increase in communication rate as measured in bits/min (100-300%), and theoretical bit rate (100-550%), while maintaining selection accuracy. Participants also overwhelmingly preferred the DS algorithms. Significance. We have developed a viable BCI algorithm that has been tested in a target BCI population which has the potential for translation to improve BCI speller performance towards more practical use for communication.