京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22310号 / 医博第4551号 / 新制||医||1040(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 渡邉 大, 教授 万代 昌紀, 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

Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome but why some brain cells and structures are initially spared by the virus is unknown. Here, a novel murine model of ZIKV infection incorporating in utero electroporation with cell type specific promotors was used to identify the time course of ZIKV infection and to determine which neural precursor cells are initially infected or spared. In vivo time course studies revealed early presence of ZIKV in apical radial glial cells (aRGCs) while infection of basal intermediate progenitor cells climbed after three days of virus exposure. ZIKV-exposed fetal brains exhibited microencephaly as early as 1 day post injection, caused by apoptosis and reduced proliferation, and this change in brain size persisted until birth regardless of developmental age at infection. During infection, 60% of aRGC basal fibers were perturbed while 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To evaluate this heterogeneous vulnerability, we generated cell type-specific fate mapping plasmid probes using a previously published single cell RNA-Seq dataset on the E15.5 mouse neocortical wall. The results indicate that one class of aRGC preferentially expresses the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than the other aRGC subtypes with low AXL expression. Together, these data highlight important temporal and cellular details of ZIKV fetal brain infection and may be important for prevention strategies and for management of congenital Zika syndrome.

Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors.

The regulation of neural precursor cells (NPCs), which encompass neural progenitor and neural stem cells (NSCs), is fundamental for proper brain development and function. These cells are regulated by orchestrated signalling within their local environment. Aberrant aspects of cell proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome (FXS)—a disorder characterized by intellectual and social changes due to the silencing of the gene encoding FMRP. The biology of hippocampal NPCs in FXS during early postnatal development has not been studied, despite high FMRP expression levels in the hippocampus at the end of the first postnatal week. In this thesis, the Fmr1-knockout (KO) mouse model was used to study hippocampal cell biology during early postnatal development. A tissue culture assay, used to study the effect of astrocyte-secreted factors on the proliferation of NSCs, indicated that astrocyte secreted factors from Fmr1-KO brains enhanced the proliferation of wild type, but not Fmr1-KO NSCs (Chapter 3). Next, the proliferation and cell cycle profiles of NPCs in vitro and in vivo studied with immunocytochemistry, Western blotting, and flow cytometry revealed decreased proliferation of NPCs in the Fmr1-KO hippocampus (Chapter 4). Finally, cells isolated from the P7 dentate gyrus and characterized by flow cytometry, showed a reduced proportion of NSCs and an increased proportion of neuroblasts—neuronal committed progenitors—in Fmr1-KO mice. Together, these results indicate that hippocampal NPCs show aberrant proliferation and neurogenesis during early postnatal development. This could indicate stem-cell depletion, increased quiescence, or a developmental delay in relation to lack of FMRP and uncovers a new role for FMRP in the early postnatal hippocampus. In turn, elucidating the mechanisms that underlie FXS will aid in the development of targeted treatments. / Thesis / Doctor of Philosophy (PhD) / Fragile X syndrome is the leading inherited cause of intellectual impairment and autism spectrum disorder. The syndrome is caused by a defect in one gene. This gene has been suggested to play a role in regulating the birth of new brain cells termed neural precursor cells. The importance of neural precursor cells stems from their ability to generate neurons and glia, the main cells in the brain. In this thesis, I focus on studying neural precursor cells from the hippocampus, a brain region important for learning and memory. A mouse model was used to compare neural precursor cells from healthy and Fragile X mice during early postnatal development. I found that neural precursor cells do not divide as much as they should in the Fragile X mouse hippocampus. The results help to determine the causes for learning and memory deficits in Fragile X and potentially open avenues for intervention.

Neurons are generated from unspecialized neural precursor cells (NPCs) in a process termed neurogenesis. This neuronal development continues throughout life in the ventricular zone (VZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. NPCs undergo a complex and highly regulated set of behaviours in order to ultimately integrate into the existing brain circuitry as fully functional neurons. Recently the pannexin (Panx) large-pore channel proteins were discovered. One family member, Panx1 is expressed in the nervous system in mature neurons, and acts as an ATP release channel in various cell types throughout the body. Post-natal NPCs are responsive to ATP via activation of purinergic receptors, which modulate a variety of NPC behaviours. I therefore investigated the hypothesis that Panx1 was expressed in post-natal VZ NPCs, where it functioned as an ATP release channel and regulated neuronal development. In the course of my studies, I found that Panx1 positively regulated NPC proliferation and migration, and negatively regulated neurite outgrowth in vitro. Using an NPC-specific Panx1 knock-out strategy, I showed that Panx1 expression was required for maintenance of a consistent population of VZ NPCs in vivo in both healthy and injured brain. Together these data indicated that Panx1 directed NPC behaviours associated with neuronal development both in vitro and in vivo. To further understand the molecular underpinnings of this regulation, I examined the Panx1 interactome, and uncovered a novel association with collapsin response mediator protein 2 (Crmp2). Functional studies suggested that this interaction likely was at least in part responsible for Panx1’s negative impact on neurite outgrowth. Overall, my results represent important novel findings that contribute to our understanding of post-natal neuronal development and the molecular function of Panx1 within the brain. / Graduate / 0317 / 0379 / leighws@uvic.ca

Hippocampal neurons are vulnerable to injury, as indicated by the prevalence of learning and memory deficits following traumatic brain injury. Research indicates that proliferation of neural precursor cells increases following brain injury, which implies that there is an endogenous response in the hippocampus to replenish neurons and restore cognitive function. Studies show that mitogenic growth factors may drive this proliferative response; one of which is epidermal growth factor. Because adults and the elderly manifest the most enduring deficits following TBI, it is critical to investigate how EGF expression following injury may relate to injury-induced cell proliferation and the degree of cognitive recovery observed with aging. In the current study, we assessed the temporal and spatial expression of EGF in the injured hippocampus with age. Our results suggest that EGF expression increases following TBI, and this increase is more significant in the younger brain. Additionally, we investigated the phenotype and localization of cells that express EGF following injury.

Schizophrenia is a major psychiatric disorder that affects 1% of the world population and is among the 10 leading worldwide causes of disability. Disrupted-In- Schizophrenia (DISC1) is one of the most studied risk genes for mental illness and is disrupted by a balanced translocation between chromosomes 1 and 11 that co-segregates with major mental illness in a single large Scottish family. DISC1 is a scaffold protein with numerous interactors and has been shown to hold key roles in neuronal progenitor proliferation, migration, cells signalling and synapse formation and maintenance. The studies herein provide the platform in order to investigate the molecular and cellular consequences of the t(1;11) translocation using induced pluripotent stem cells (iPSCs)-derived neural precursor cells and neurons from within-family carriers and controls. Towards this end, several iPSC lines have been converted into neural progenitor cells (NPCs) and differentiated into physiologically active forebrain neurons following well-characterised protocols. These cells were characterised in terms of basic marker expression at each developmental stage. Inter-line variation was observed in all subsequent experiments but overall t(1;11) lines did not generate less neuronal or less proliferating cells compared to control lines. Furthermore, the expression pattern of genes disrupted by the t(1;11) translocation was investigated by RT-qPCR. DISC1 was reduced by ~50% in the translocation lines, both neural precursors and neurons. This observation corresponds to previous findings in lymphoblastoid cell lines (LBCs) derived from members of the same family. Moreover, DISC1 expression was found to increase as neural precursors differentiation to neurons. Two other genes are disrupted by the t(1;11) translocation;DISC2 and DISC1FP1. Their expression was detectable, but below the threshold of quantification. Similarly, DISC1/DISC1FP1 chimeric transcripts corresponding to such transcripts previously identifies in LBCs from the family were detectable, but not quantifiable. A fourth gene, TSNAX, was also investigated because it is located in close proximity to, and undergoes intergenic splicing with, DISC1. Interestingly, TSNAX was found to be altered in some but not all time points studied, in the translocation carriers compared to control lines. In addition to breakpoint gene expression profiling, iPSC-derived material was used to investigate neuronal differentiation. There seemed to be attenuation in BIII-TUBULIN expression at two weeks post-differentiation, while NESTIN, MAP2 and GFAP expression was similar between translocation carrier and control lines at all time points studied. I also had access to targeted mice designed to mimic the derived chromosome 1 of the t(1;11) balanced translocation. Using RT-qPCR Disc1 expression was found to be 50% lower in heterozygous mice compared to wild types, and I detected a similar profile of chimeric transcript expression as detected in translocation carrier-derived LBCs. These observations support my gene expression studies of the human cells and indicate that the iPSC-derived neural precursors and neurons can be studied in parallel with the genome edited mice to obtain meaningful insights into the mechanism by which the t(1;11) translocation confers substantially elevated risk of major mental illness. In conclusion, the studies described in this thesis provide an experimental platform for investigation of the effects of the t(1;11) translocation upon function and gene and protein expression in material derived from translocation carriers and in brain tissue from a corresponding mouse model.

Adult neural precursor cells (NPCs) are a heterogeneous population of mitotically active, self-renewing multipotent cells of both adult and developing CNS. They can be expanded in vitro in the presence of mitogens. The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). A stereological count demonstrates that mice with significant cell loss exhibit highest survival of grafted NPCs and migration to the vicinity of PCs as compared to wt and younger grafted animals. These animals showed improved motor skills as compared to sham animals. Confocal analysis and profiling shows that many of implanted cells present in the cerebellar cortex have formed gap junctions with host PCs and express connexin43. Grafted cells did not adopt characteristics of PCs, but stereological and morphometric analysis of the cerebellar cortex revealed that grafted animals had more surviving PCs and a better preserved morphology of these cells than the control groups. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. In this study, evidence for a neuroprotective effect came, in addition to motor behavior improvement, from stereological and electrophysiological analyses and suggest that timing of stem cell delivery is important to determine its therapeutic effect. In a brain stem cell niche, NSCs reside in a complex cellular and extracellular microenvironment comprising their own progeny, ependymal cells, numerous blood vessels and various extracellular matrix molecules. Recently, it was reported that blood vessel ECs-NSCs crosstalk plays an important role in tissue homeostasis. Bloodstream offers a natural delivery vehicle especially in case of diffuse neurodegenerative diseases which require widespread distribution of exogenous cells. As NSCs are confronted with blood-brain barrier endothelial cells (BBB-ECs) before they can enter into brain parenchyma, we investigated their interaction using primary cultures in an in vitro BBB model. We isolated human fetal neural precursor cells (hfNPCs) from aborted fetal brain tissues and expanded in vitro. We showed that in an in vitro model, human BBB endothelium induces the rapid differentiation of hfNPCs and allows them to cross the endothelial monolayer, with the differentiated progeny remaining in close contact with endothelial cells. These results are not reproduced when using a non-BBB endothelium and are partly dependent on the cytokine MCP1. Our data suggest that, in the presence of attractive signals released by a damaged brain, intravascularly administered NPCs can move across an intact BBB endothelium and differentiate in its vicinity. Overall, our findings have implications for the development of cellular therapies for cerebellar degenerative diseases and understanding of the brain stem cell niche.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

<p>Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis [1;2]. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings [3]. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants (termed the glial scar), making the biocompatibility of implanted electrodes a primary concern in device design. Previous studies have focused on modifying the form factor of recording arrays, implanting such arrays in experimental animals, and, upon explantation, evaluating the glial scarring in response to the implant after several weeks in vivo. Because of a lack of information regarding the mechanisms involved in the tissue reaction to implanted biomaterials in the brain, it is not surprising that these in vivo studies have met with limited success. This dissertation describes the development of a simple, controlled in vitro model of glial scarring and the utilization of that model to probe the cellular and molecular mechanisms behind glial scarring.</p><p>A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease [4]. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-D-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 mm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording. </p><p>This initial model recreated many of the hallmarks of glial scarring around electrodes used for recording in the brain; however, the model lacked the reproducibility necessary to establish a useful characterization tool. After the protocol was amended to resemble protocols typically used to culture neural stem/precursor cells, an intense scarring reaction was consistently seen [5]. To further optimize and characterize the reaction, six independent cell culture variables (growth media, seeding density, bFGF addition day, serum concentration in treatment media, treatment day, and duration of culture) were varied systematically and the resulting scars were quantified. The following conditions were found to give the highest level of scarring: Neurobasal medium supplemented with B27, 10% fetal bovine serum at treatment, 10 ng/ml b-FGF addition at seeding and at treatment, treatment at least 6 days after seeding and scar growth of at least 5 days. Seeding density did not affect scarring as long as at least 500,000 cells were seeded per well, but appropriate media, bFGF, and serum were essential for significant scar formation. </p><p>The optimized in vitro model was then used to help uncover the underlying molecular and cellular mechanisms behind glial scarring. A microwire coating that mimics the basal lamina present within glial scars was developed that allows cells responding to the coated microwire to be isolated and evaluated (i.e. through cell counting or cell staining). A panel of soluble factors known to be involved in glial scar formation was added to the media and the cellular response was recorded. The extent of cell accumulation on the coated microwires was significantly increased by titration of the culture with serum, the pleotropic growth factor bFGF, the inflammatory cytokines IL-1&alpha; and IL-1&beta;, and the growth factors PDGF and BMP-2. The other fourteen soluble factors tested had little to no effect on the number of cells that attached to the coated microwires, although a specific blocker of the bFGF receptor was able to abrogate the effect of bFGF. This study proposes essential roles in glial scarring of serum, which infiltrates brain tissue upon disruption of the blood-brain barrier, and bFGF, which is a necessary growth and survival factor for the neural precursor cells that respond to injury. These insights suggest repeated rounds of implant micromotion-induced cellular damage, with the resultant neuronal death, serum release, and bFGF deposition may thicken the glial scar and lead to recording signal loss.</p> / Dissertation