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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23813号 / 医博第4859号 / 新制||医||1059(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 中本 裕士, 教授 花川 隆, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Microglia Purinergic Receptor-Mediated Neuroinflammation In Alzheimer’s Disease Neurodegeneration involves a complicated cascade of homeostatic dysfunction that converges on neuron loss and cognitive decline, involving complex immune, metabolic, and cell cell crosstalk pathways. The complicated interplay and heterogeneous nature of these factors in the brain make therapeutic development challenging. Recent advances have placed the immune system as an important driver of neurodegeneration both mechanistically and genetically. Microglia are the professional phagocytes that inhabit the brain and direct these inflammatory pathways, which can have reparative or destructive outcomes on the brain parenchyma. While various genetic risk factors for neurodegeneration reside in microglia, how these trigger and facilitate disease requires further investigation. In the present dissertation, I investigate inflammatory activation in microglia upon various damage or pathology-associated stimuli by utilizing a primary human monocyte-derived microglia-like cell (MDMi) model from a diverse donor cohort, which allows for the examination of genetically driven differences. I find that MDMi stimulated through ATP-mediated P2RX7 activation display reduced phagocytic function for amyloid beta uptake, and this pathway is also influenced by individual donors’ SPI1 genotype which has been associated with Alzheimer’s disease in previous computational studies. These experiments demonstrate functional outcomes related to AD genetics in immune cells. Previous computational studies have identified cognitive-decline associated gene modules expressed in human brain tissues from late-stage AD. I conducted in vitro follow up experiments to interrogate these genetic findings which is crucial for validating RNA sequencing data in a biological model. To interrogate differential MDMi inflammatory pathways, I treated cells with the toxic immunostimulatory molecule lipopolysaccharide (LPS), or its non-toxic derivative monophosphoryl lipid A (MPLA) which has positive immune properties currently utilized in vaccine adjuvants. My results indicated that individual gene expression in this module does not shift in a uniform manner upon LPS or MPLA challenge, suggesting more nuanced in vitro interrogation is required to identify conditions propagating this end stage disease phenotype. Microglia serve as the primary immune cells of the brain but also interact closely with astrocytes, large glial cells that facilitate neuronal homeostasis and are central players in AD due to their high apolipoprotein (APOE) production. Given the newly appreciated role of cellular crosstalk in neurological disease pathogenesis, I sought to optimize a protocol for isolation of primary mouse astrocytes for coculture with MDMi and investigation of non-direct cell contact interactions through astrocyte supernatants. Described in this dissertation is my optimized protocol for purified mouse astrocyte isolation from mice expressing humanized APOE2, APOE3, or APOE4. By developing this model, I was able to discern differential changes to MDMi gene expression in the presence of APOE2, 3, or 4 astrocyte supernatants. Verification of these tools allows further exploration of APOE genotype on glial crosstalk and downstream AD pathology. Overall, this work uncovers important mechanisms of human microglia activation through AD genetics and extracellular P2RX7 receptor behavior. By interrogating these scientific questions in a human microglia model derived from donors of various genetic and age backgrounds, we can assess how real biological variation modulates canonical inflammatory pathways. This adds powerful clinical relevance as AD and other neurodegenerative conditions can present a very heterogenous phenotype pathologically and therefore may require the nuance of more personalized medicine therapeutically.

The goal of this research study was to create an artificial annulus fibrosus similar to that of the natural intervertebral disc, as well as find preliminary results for vertebral endplate connection and nucleus pulposus internal pressure, for the correction of disc degeneration in the spine. The three-part composite samples needed to demonstrate good shock absorption and load distribution while maintaining strength and flexibility, and removing the need for metal in the body, something of which no current total disc replacement or spinal fusion surgery can offer. For this study, the spinal disc was separated into its three different components, the annulus fibrosus, the nucleus pulposus, and the vertebral endplates, each playing a vital role in the function of the disc. Two low-cost materials were selected, a Covestro polyurethane and cellulose nanocrystals, for the purpose of creating a polymer composite spinal disc implant. A methodology was established for creating the cast composite material for use as an annulus fibrosus, while also investigating its mechanical properties. The same composite material was used to acquire preliminary results for vertebral endplate connection to the synthesized annulus, however no additional material was used to determine or mimic the mechanical properties of these endplates, due to time constraints. Also because of time constraints, the nucleus used in this study was only comprised of water with no other additives for preliminary testing since the natural nucleus is comprised of about 80-90% water. These properties were then compared to the mechanical properties of the natural disc, so that they could be finely tuned to emulate the natural disc. It is shown in this study that the composite material, when swelled in water, was able to mimic the annulus fibrosus in tensile strength and modulus, however showed higher compressive strength and modulus than ideal. The samples also did not undergo any permanent deformation within the realm of force actually introduced to the natural disc. The vertebral endplates showed decent adhesion to the synthesized annulus, however there were slight defects that became failure concentrators during compression testing. The nucleus showed promising results maintaining good internal pressure to the system causing better compressive load distribution, with barreling of the samples. / Master of Science / Spinal disc degeneration is a very prevalent problem in today’s society, effecting anywhere from 12% to 35% of a given population. It usually occurs in the lumbar section of the spine, and when severe enough, can cause bulging and herniation of the intervertebral disc itself. This can cause immense lower back pain in individual’s stricken with this disease, and in the US, medical costs associated with lower back pain to exceed $100 billion. Current solutions to this problem include multiple different treatment options of which, spinal fusion surgery and total disc replacement (TDR) are among the most common. Although these treatments cause pain relief for the majority of patients, there are multiple challenges that come with these options. For example, spinal fusion surgery severely limits the mobility of its patients by fusing two vertebrae together, disallowing any individual movement, and TDR can cause hypermobility in among the vertebrae and offer little to no shock absorption of loads. Therefore, a better treatment option is needed to relieve the pain of the patients, as well as maintain equal motion, shock absorption, and load cushioning to that of the normal intervertebral disc and remaining biocompatible. The goal of this research study was to create a three-component system, like that of the natural intervertebral disc, for the use of spinal disc replacement and to replace current options. The fabricated system was comprised of the three components found in the natural intervertebral disc; the annulus fibrosus, the nucleus pulposus, and the vertebral endplates. Because the system will need to go in-body, the materials used were all characterized as biocompatible materials; the polyurethane currently being used in medical devices and implants, and the cellulose nanocrystals (CNCs) coming from natural cellulose in sources such as wood and plants. The results determined that the mechanical properties of the system can be fine-tuned in order to mimic the natural strength and cushioning capabilities of the natural disc, based on CNC content added to the polyurethane, and when all three components of the system are added together, the compressive stress-strain is most similar to the natural disc in compression. However, the system did show failure in the connection between the annulus fibrosus and vertebral endplates, causing herniation of the nucleus similar to the initial problem attempting to be solved. For this, more ideal fabrication methods should be researched in the future including 3D printing techniques, injection molding, and roll milling. As well as alternate fabrication techniques, cell grow and viability should be determined to show that cells don’t die once the system in implanted.

T-cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this dissertation aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2, but also weakly at RRM3, and Zn2+ binds primarily to RRM3. The binding of Zn2+ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn2+ caused the TIA1:DNA complexes to collapse, indicating that Zn2+ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3 and introducing a mesh of crosslinks in the supramolecular complex. Furthermore, this dissertation presents an investigation into the interdomain interactions between RRM2 and RRM3, facilitated by the successful preparation of segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn2+ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn2+ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.