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Fused in Sarcoma (FUS) is a nuclear RNA binding protein functioning in a number of essential cellular processes such as RNA processing and DNA damage response. Mutations in FUS gene contribute to 5% of familial Amyotrophic Lateral Sclerosis (ALS) characterized by FUS protein cytoplasmic aggregation. Despite efforts have been made in the past decade, mechanisms of FUS aggregates to induce cytotoxicity are not fully understood. In addition, wild-type FUS protein has been found mis-localized to cytoplasm in sporadic ALS and Frontotemporal Dementia (FTD) patients with unclear mechanisms. Here, we aimed to address the functional consequences of ALS mutant FUS aggregation and investigate the mechanisms of wild-type FUS cytoplasmic translocation. This dissertation is divided into three parts: In the first part, we review pathophysiological mechanisms of FUS and other ALS mutant genes which induce cell death via disrupting six major cellular processes: mRNA processing, non-sense mediated decay, mitochondrial functions, nucleocytoplasmic transport, autophagy and DNA damage response. In the second part, we aimed to understand the functional consequences of RNA sequestration by FUS aggregates. We performed RNA immunoprecipitation against exogenous or endogenous FUS in the transfected cell lines and mutant FUS ALS patient fibroblasts to isolate RNAs associated with wild-type or ALS mutant FUS. Next, we analyzed the isolated RNAs using poly(A+) RNA-specific sequencing 3’READS and RT-qPCR, and we found many nuclear-encoded respiratory chain complex mRNAs are top-enriched transcripts associated with ALS mutant or overexpressed wild-type FUS. We further demonstrated that respiratory chain complex mRNAs are sequestered in mutant FUS cytoplasmic aggregates and the encoded protein expression levels are suppressed. Finally, we showed that knockdown of respiratory chain complex proteins encoded by FUS-sequestered transcripts can recapitulate mitochondrial dysfunction observed in FUS-transfected cell lines. Our findings in the second part thus provides a novel mechanism by which ALS mutant FUS, as well as overexpressed wild-type FUS, to induce mitochondrial dysfunction via preferential sequestration of respiratory chain complex mRNAs. The third part focuses on understanding pathways affecting FUS nucleocytoplasmic distribution. By using pharmacological treatments and immunofluorescence, we found that nuclear RNA transcription, export and decay substantially modulate nucleocytoplasmic distribution of wild-type FUS protein. Moreover, we report that FUS antibodies used in immunofluorescence significantly affect the results of nucleocytoplasmic ratio quantification. Intriguingly, we observed altered serine-2/-5 phosphorylation on RNAPII CTD as well as reduced number of nascent transcripts in sporadic ALS patient cells, indicating aberrant transcriptional activity related to cytoplasmic accumulation of nuclear RNA binding proteins. Our findings in the third part provide insights to the importance of nuclear RNA metabolism in modulating FUS localization. We also addressed the inconsistent results reported in previous studies regarding FUS nucleocytoplasmic distribution in response to stress. Altogether, these findings suggest proof-of-principle mechanisms of FUS toxic function and aberrant localization linked to ALS and FTD disease spectrum.

An electroencephalographic-based brain-computer interface (BCI) can provide a non-muscular method of communication. A general model for P300-based BCI stimulus presentations is introduced - the "m choose n" or C(m (number of flashes per sequence), n (number of flashes per item)) paradigm, which is a universal extension of the previously reported checkerboard paradigm (CBP). C(m,n) captures all possible (unconstrained) ways to flash target items, and then applies constraints to enhance ERP's produced by attended matrix items. We explore a C(36,5) instance of C(m,n) called the "five flash paradigm" (FFP) and compare its performance to the CBP. Eight subjects were tested in each paradigm, counter-balanced. Twelve minutes of calibration data were used as input to a stepwise linear discriminant analysis to derive classification coefficients used for online classification. Accuracy was consistently high for FFP (88%) and CBP (90%); information transfer rate was significantly higher for the FFP (63 bpm) than the CBP (48 bpm). The C(m,n) is a novel and effective general strategy for organizing stimulus groups. Appropriate choices for "m," "n," and specific constraints can improve presentation paradigms by adjusting the parameters in a subject specific manner. This may be especially important for people with neuromuscular disabilities.

BCI can provide communication for people locked in by amyotrophic lateral sclerosis (ALS). Empirical examination of how disease progression affects brain-computer interface (BCI) performance has not been investigated. This pilot study uses a longitudinal design to investigate changes in P300-BCI use as ALS disability increases. We aimed to (a) examine the relationship between BCI accuracy and the ALS/Functional Rating Scale and (b) examine changes in the event-related potential (ERP) components across time. Eight subjects have been enrolled in the study. BCI accuracy was measured and ERP components were assessed by a principal component analysis (PCA). Two subjects have been followed for an average of nine-months, and BCI accuracy is 99.6%. While many research obstacles remain, these preliminary data help elucidate the relationship between BCI performance and disease progression.

Our objective was to develop and validate a new brain-computer interface (BCI) system suitable for long-term independent home use by people with severe motor disabilities. The BCI was used by a 51-year-old male with ALS who could no longer use conventional assistive devices. Caregivers learned to place the electrode cap, add electrode gel, and turn on the BCI. After calibration, the system allowed the user to communicate via EEG. Re-calibration was performed remotely (via the internet), and BCI accuracy assessed in periodic tests. Reports of BCI usefulness by the user and the family were also recorded. Results showed that BCI accuracy remained at 83% (r -.07, n.s.) for over 2.5 years (1.4% expected by chance). The BCI user and his family state that the BCI had restored his independence in social interactions and at work. He uses the BCI to run his NIH-funded research laboratory and to communicate via e-mail with family, friends, and colleagues. In addition to this first user, several other similarly disabled people are now using the BCI in their daily lives. In conclusion, long-term independent home use of this BCI system is practical for severely disabled people, and can contribute significantly to quality of life and productivity.

A brain-computer interface is a device that uses signals recorded from the brain to directly control a computer. In the last few years, P300-based brain-computer interfaces (BCIs) have proven an effective and reliable means of communication for people with severe motor disabilities such as amyotrophic lateral sclerosis (ALS). Despite this fact, relatively few individuals have benefited from currently available BCI technology. Independent BCI use requires easily acquired, good-quality electroencephalographic (EEG) signals maintained over long periods in less-than-ideal electrical environments. Conventional, wet-sensor, electrodes require careful application. Faulty or inadequate preparation, noisy environments, or gel evaporation can result in poor signal quality. Poor signal quality produces poor user performance, system downtime, and user and caregiver frustration. This study demonstrates that a hybrid dry electrode sensor array (HESA) performs as well as traditional wet electrodes and may help propel BCI technology to a widely accepted alternative mode of communication.

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) now appears to be a cause of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). Its production by cyanobacteria throughout the world combined with multiple mechanisms of BMAA neurotoxicity, particularly to vulnerable subpopulations of motor neurons, has significantly increased interest in investigating exposure to this non-protein amino acid as a possible risk factor for other forms of neurodegenerative illness. We here provide a brief overview of BMAA studies and provide an introduction to this collection of scientific manuscripts in this special issue on BMAA.

The accumulation of misfolded proteins within intracellular aggregates is a distinctive feature observed within multiple neurodegenerative diseases (NDDs). However, the genes and pathways that regulate protein misfolding, aggregation, and subsequent cellular toxicity remain poorly understood. Here I describe a high-throughput discovery platform that enables the simultaneous screening of dozens of neurodegenerative disease models to rapidly uncover genetic modifiers that alter the solubility and toxicity of a wide variety of aggregation-prone proteins. From these studies, I identify the human HSP40 chaperone, DNAJB6 as a potent rescuer of the misfolding and proteotoxicity of multiple RNA-binding proteins implicated in Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) including FUS, TDP-43, and hnRNPA1. I, with collaborator help, further demonstrate that DNAJB6 has an intrinsic ability to phase separate under physiologic conditions and can alter the properties of FUS containing condensates by maintaining them in a gel-like state over long periods, preventing FUS aggregation. By conducting domain mapping studies and a deep mutational scan on DNAJB6, I am able to gain detailed insight into its mechanism of action while also uncovering a series of novel variants with enhanced activity. During the development of this multiplexed screening approach for neurodegenerative disease models, research was interrupted by a global pandemic caused by SARS-CoV-2. I realized that the themes of studying proteotoxicity of multiple related, yet distinct models could be applied towards drug development to identify inhibitors of the essential 3CL proteases encoded by multiple coronaviruses, which cause proteotoxicity when expressed in cells. As such, I develop and describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This essay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, I identify compounds with broad 3CLpro inhibitory activity. I also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. I observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. I identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses.

Final publication is available at http://www.sciencedirect.com/science/article/pii/S1873506115001191 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19579号 / 医博第4086号 / 新制||医||1013(附属図書館) / 32615 / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

SCIENTIFIC REPORTS へのhyperlink http://www.nature.com/articles/srep19118 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19926号 / 医博第4146号 / 新制||医||1017(附属図書館) / 33012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 井上 治久, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM