京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13163号 / 論医博第2150号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 渡邉 大, 教授 高橋 淳 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive memory loss, brain atrophy, and eventual death. AD pathology is characterized by the accumulation of neurotoxic amyloid-beta (Aβ) plaques, synapse loss, neurofibrillary tangles (NFTs), and neurodegeneration. The APOE4 allele is associated with a 3-fold increased risk for AD and results in increased Aβ plaque deposition, reduced Aβ clearance, and reduced synaptic plasticity. Although APOE expression is upregulated in microglia in AD, APOE is expressed primarily by astrocytes in the CNS. It is not well understood how astrocytic APOE drives the mechanisms that result in worsened AD outcomes. Here, digital spatial profiling and bioinformatics data suggest that APOE4 causes transcriptional dysregulation in early AD and may disrupt neuronal processes via astrocytes. Whole transcriptome data from plaque and non-plaque regions in the cortices and hippocampus of 4- and 8-month-old AD model mice expressing humanized APOE4/4 or APOE3/3 (control) were analyzed. Transcriptional dysregulation was increased in APOE4/4 AD mice compared to that in APOE3/3 at 4 but not 8 months of age, suggesting that early dysregulation of APOE4-driven disease mechanisms may shape degenerative outcomes in late-stage AD. Additionally, APOE4/4 potentially functions via plaque-independent mechanisms to influence neuronal function in early AD before the onset of pathology. Single-nuclei RNA sequencing data were obtained from human post-mortem astrocytes and the bioinformatic analyses revealed a novel astrocyte subtype that highly expresses several top genes involved in functional alterations associated with APOE4, including neuronal generation, development, and differentiation, and synaptic transmission and organization. Overall, our findings indicate that APOE4 may drive degenerative outcomes through the presented astrocyte candidate pathways. These pathways represent potential targets for investigations into early intervention strategies for APOE4/4 patients. / 2024-05-22

The objective of the completed research was to characterize the morphology of individual neurons within monolayer networks of fetal mouse spinal tissue via intraperikaryal injections of horseradish peroxidase (HRP). Thirty labelled neurons were reconstructed via camera lucida drawings and morphometrically analyzed.

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Early growth response gene – 1 (Egr1) encodes a protein widely present in mammalian body, such as connective tissue, cardiac tissue, the liver, and the brain. As a transcription factor (TF), it is involved in processes that take place in the endocrine, digestive, immune, musculo-skeletal and central nervous systems, for instance, B cell maturation upon B cell receptor activation, tendon repair upon mechano-stimulation, and long-term spatial memory formation. In mammalian brains, EGR1 controls the responses to environmental stimuli such as chronic stress and physical contact. It also participates in processes such as long-term memory consolidation and synapse re-structuring. It plays a role in enacting responses and qualities of gene transcription cascades upon neuronal stimulation. Inside the epigenetic realm, EGR1 recruits Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) to remove DNA methylation at target loci. Due to its critical functions during brain development and upon neuronal activation, mis-regulation of EGR1 is associated with neuropsychological disorders such as post-traumatic stress disorder (PTSD) and schizophrenia (SCZ) in humans. In this study, we performed bioinformatics analysis with brain methylomes and predicted EGR1 may interact with myocyte enhancer factor 2C (MEF2C), which is known to be involved in many similar processes as EGR1, such as synapse architecture, cell migration, and learning and memory. EGR1 and MEF2C ChIP-seq data derived from mouse frontal cortex suggest these two proteins may regulate a common set of downstream genes. To begin, co-immunoprecipitation experiments were performed with HEK293T cells co-transfected with EGR1-FLAG and MEF2C-HA tagged constructs, allowing for specific interaction identification without endogenous protein expression interference. Furthermore, co-immunoprecipitation experiments performed with brain tissues additionally indicated the two proteins interact with each other endogenously. Altogether, this study provides protein-protein interaction evidence for EGR1 and MEF2C in cultured HEK293 cells and in the cortices of adult male mice. This information provides a foundation for future examinations of how these two TFs interact to initiate cascading events following neuronal stimulation. / Master of Science / Early growth response gene – 1 (EGR1) encodes a protein that can be found in animals such as fruit flies, mice, rats, and humans. In mammals, it is widely expressed in the cardiovascular, endocrine, digestive, immune, musculo-skeletal and central nervous systems (CNS). Within the CNS, EGR1 is known as an essential transcription factor involved in brain development. More specifically, EGR1 plays a role in how the early brain develops in response to environmental stimuli, formation of synapse architecture and certain types of memories. Many gene networks involved in growth and development rely on EGR1 to regulate functions such as synapse reformation after exposure to the environment. EGR1 is known to have numerous partners with whom it interacts to execute its functions. It is also involved in epigenetic regulation, which is a process by which genes are silenced or activated without changing DNA sequences in the genome. EGR1 may directly interact with TET1 to demethylate EGR1 target sites in the genome, and to increase gene transcription. In memory development, EGR1 plays a key role ensuring short-term auditory fear memory can be converted to long-term memory, and also ensures long-term spatial memory. In this study, our computational analyses suggest that EGR1 may interact with MEF2C. This work provides evidence of a protein-protein interaction of EGR1 and MEF2C in cultured cells and in the brain cortical areas of mice. Such an interaction may explain why these two genes regulate overlapped biological processes within the brain and sheds lights on how cascading events are initiated following neuronal stimulation.

Yes / Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. / Funded by a biomedical research grant from the Motor Neurone Disease Association (ref Rattray/Apr15/837-791). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease, which, before the approval of therapies, was the leading genetic cause of infant mortality. The primary features of this pathology are progressive muscle weakness and atrophy, due to the degeneration of α-motor neurons in the anterior horn of the spinal cord. SMA is caused by deletions or mutations in the Survival Motor Neuron gene (Smn1), which induce reduced levels of the SMN protein. Since 1999, this disease has been primarily associated with splicing defects caused by loss of SMN protein due to its role in ribonucleoparticle biogenesis. However, further research revealed that this mechanism alone is not sufficient to explain the pathogenesis of the disease. More recent findings revealed that deficient SMN levels lead to defective translation in primary motor and cortical neurons, and in multiple tissues at the late stage of disease in the severe Taiwanese mouse model of SMA. Furthermore, SMN protein has been confirmed to be a ribosome-associated protein in vitro, in mouse cell lines and in vivo, and to physiologically regulate the translation of a particular subset of transcripts (defined as SMN-specific transcripts), which are characterized by specific sequence features. Upon SMN loss, the translation of this subset of transcripts is defective. SMN protein is ubiquitously expressed and its levels vary at different developmental stages and tissues in physiological conditions, leading to the hypothesis that translational defects may vary accordingly. However, the effect of SMN loss on translation across different tissue types, SMA mouse models, and disease stages is yet to be clarified. To investigate the link between SMN loss and translational defects in SMA, I took advantage of ribosome profiling to obtain the translatome from multiple tissues, stages and disease mouse models. Given that SMN is ubiquitously expressed, brain, spinal cord and liver were collected to investigate if common features underly translational defects upon its loss in these tissues. Since little is known about how translational impairments are modulated over time, tissues were collected from various developmental and disease stages, ranging from the embryo to the post-natal early-symptomatic stage of SMA. Furthermore, translation defects were studied in multiple models of SMA have ranging from severe to mild (i.e., Taiwanese, Delta7 and Smn2b/-), allowing for the exploration of the heterogeneity of the SMA clinical phenotype. Hence, the tissues were collected from three SMA mouse models (i.e., Taiwanese, Delta7, and Smn2b/-), allowing for the investigation of translational impairments in conditions that range from severe to mild SMA. A wide range of computational approaches was adopted to analyze ribosome profiling data from multiple perspectives, including Principal Component Analysis (PCA), pipelines for the analysis of RiboSeq positional information, differential and Gene Ontology enrichment analysis, and network methodologies. This set of tools applies to the study of ribosome profiling data and allows to investigate the translational mechanisms underlying SMA. This multilevel analysis holds difficulties in the representation and interpretation of the obtained results due to the number of variables (i.e., tissue, stage, model, and disease condition). I hence developed an R package to support the visualization of changes occurring in omics data from complex experimental designs. Next, I focused on the identification of translational defects in SMA through pairwise differential analyses performed on each set of experiments. This allowed me to identify significantly altered transcripts within each comparison. Despite poor overlaps between the sets of translationally dysregulated transcripts across the different stages, tissues, and models, commonly enriched biological processes were found. The analysis of sequence features on translationally dysregulated transcripts across all the stages, tissues, and models revealed the presence of features similar to those already found on the SMN-specific transcripts. In addition, based on network methodologies, I investigated the system-wide effects of SMN loss on connectivity patterns at the translational level, by taking advantage of network-based methodologies to integrate all sets of experiments and unravel any relationships between genes at the translatome level. Causal-inference networks, coupled with differential network analysis, complemented the standard differential analysis by modeling how the fluctuations in reciprocal transcript-specific ribosome occupancy might influence each other. This allowed to detect disrupted relationships in the disease condition across the multiple tissues, stages and models. In summary, this thesis provides, to my knowledge, the first multi-tissue, -stage, and -model translatome analysis to investigate the mechanisms underlying SMA. Furthermore, results provided within this work confirm that translation dysregulation is a common feature of SMA pathology across multiple tissues, stages, and SMA models. This highlights that the presence of specific sequence features of translationally dysregulated transcripts is a common link between defective translational regulation and SMN loss. Moreover, the detection of disrupted connectivity patterns at the translatome level underlies that a strong remodeling occurs upon SMN loss, and further emphasizes the pivotal role of this protein in translation. These outcomes highlight the importance of further investigating the mechanisms underlying defective translation in SMA from a system perspective to provide a comprehensive understanding of this pathology and promote the development of effective therapeutic strategies.