The main goal of this study was to further explore the hypothesis that experience-dependent neural network activity and neurotransmission can modulate adult OPC proliferation and differentiation. More specifically, we used stereology to establish whether extensive reference memory training and system-wide administration of GABAergic agonists and antagonists could influence the proliferation and differentiation of adult OPCs, as well as the prevalence of OPC-neuron pairs. Analysis of the effects of reference memory training on OPC proliferation and differentiation corresponds to experiment 2, analysis of the effects of GABAergic agents on OPC proliferation and differentiation corresponds to experiment 3, and analysis of the effects of both reference memory training and GABAergic agents on OPC-neuron pairs, as well as an histological analysis of these closely apposed cells, corresponds to experiment 4.

A key problem in multiple sclerosis (MS) is the diminished capacity for myelin repair. Although oligodendrocyte (OL) precursors can be seen at the lesion site, their ability to differentiate appears inhibited. MicroRNAs are key regulators of OL differentiation, and have been observed to be misregulated in MS lesions compared to healthy white matter. Thus, aberrant microRNA expression in MS lesions may disrupt the ability of incoming oligodendrocyte progenitor cells (OPC s) to differentiate. Specifically, a microRNA known as miR - 145 is downregulated as OPCs progress to OLs, but is found at unusually high levels in MS lesions. In this study, we investigated how misregulation of miR - 145 affects OL differentiation in vitro. Bioinformatic analysis revealed that putative targets of miR - 145 are significantly enriched for factors which promote actin cytoskeleton organization and myelination. An immortalized OL cell line was transduced with an inducible lentivirus to create stable lines that overexpress miR - 145. These stable lines were characterized while proliferating, early in differentiation and late in differentiation. Immunofluorescence was used to quantify changes in proliferation rate, apoptosis, branching ability and myelin gene expression. qPCR arrays were used to quantify changes in microRNA target expression levels between induced and uninduced cells. Two stable lines were created: ON - 145 - 1 and ON - 145 - 2, which upon induction, over - express miR - 145 ~33 - fold and ~11 - fold, respectively. When proliferating, no significant morphological differences nor target expression differences could be detected between induced and uninduced cells. Proliferation was significantly decreased in ON - 145 - 1 induced cells, but not in ON - 145 - 2. No changes in apoptosis frequency were detected. In contrast, during early and late differentiation, both induced cell lines showed significant morphological defects characterized by a reduction in both iii primary and secondary branching. Further, significant differences in branching ability were observed between induced cells of ON - 145 - 1 and ON - 145 - 2, suggesting a dose - dependent response to miR - 145 overexpression. Expression of MAG, a myelin marker, was also significantly lowered in induced cells of both cell lines. Finally, we found that multiple miR - 145 targets involved in promoting cytoskeletal organization and myelination were significantly decreased both early and late in differentiation. These results suggest that overexpression of miR - 145 during OL differentiation may disrupt actin organization and myelin gene expression required for successful process extension and subsequent myelinating ability. Thus, the increase in miR - 145 in MS lesions may be a significant contributing factor to the loss of myelin repair in MS lesions.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22040号 / 医博第4525号 / 新制||医||1038(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 高橋 淳, 教授 伊佐 正, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Adolescence is a period of time when the brain undergoes profound development. Myelination is a maturational process in which oligodendroglia project out lipid-rich ramifications which wrap and insulate axons. This is crucial for effective neurotransmission between brain regions and, if compromised by pharmacological insults such as alcohol, can have long-term implications on behavior and cognition. We have previously shown that adolescent alcohol impacts males and females differentially, however it remains unknown how alcohol impacts oligodendroglia during development. The goal of this study was to determine the cellular dynamics of the oligodendroglia in male and female mice through development with and without alcohol exposure. Our results suggest that sexually dimorphic temporal dynamics exist within oligodendroglia. Specifically, this population of cells is notably dynamic during adolescence in males while stable in females. In addition, preliminary studies show that alcohol may cause a restriction on differentiation of oligodendroglia in male but not female mice. Further understanding of sex differences in the mechanisms of alcohol-induced change to oligodendroglia development could create the foundation for targeted, specific therapeutic agents and allow for individualized treatment of patients suffering from alcohol use disorders and potentially other addictions.

Oligodendrocytes are predominately found in white matter of the brain, but also populate gray matter regions. Although commonly known to provide myelination of neuronal axons, these cells serve numerous other functions in the brain. A unique property of oligodendrocytes is their inherent susceptibility to oxidative stress because of several biochemical characteristics of these cells, including a high concentration of iron, high metabolic rate, and low antioxidant enzyme activity. Oxidative stress conditions are produced by inflammation, and both inflammation and oxidative stress are highly associated with major depressive disorder (MDD). Hence, the study of oligodendrocytes in the brain in MDD readily provides access to molecular mechanisms engaged by oxidative stress conditions that putatively contribute to the etiology of MDD. My laboratory studied oligodendrocytes, and other white matter cells, from postmortem tissue collected from brain donors that died as a result of suicide and other causes, focusing on those donors who had at the time of death either MDD or no psychiatric or neurologic diagnosis (controls). White matter oligodendrocytes or whole white matter in limbic brain from MDD/suicide donors demonstrated indices of elevated oxidative damage, including increased DNA oxidation, shortened telomere DNA, reduced expression of antioxidant enzyme genes, and upregulated DNA base excision repair enzymes. These abnormalities were either not observed or were only modestly evident in astrocytes collected from white matter of the same MDD/suicide donors. To determine whether this oxidative damage was restricted to white matter in the limbic brain, oligodendrocytes were captured from three other brain regions, prefrontal cortical (BA 10) white matter, occipital cortical white matter, and gray matter in the region of the brainstem locus coeruleus. Shortened telomeres and reduced expression of antioxidant enzyme genes were observed in oligodendrocytes from these additional brain regions in MDD/suicide. Since this oligodendrocyte pathology was not anatomically restricted to the limbic brain, it may be difficult to understand how it is relevant to the biological basis of emotional behaviors that are specifically associated with MDD or suicide. However, the oligodendrocyte is highly susceptible to oxidative stress; hence, the oligodendrocyte can be viewed as a “canary in the coal mine” for detecting oxidative damage to the brain. Therefore, elucidation of the molecular pathways activated by oxidative damage in these cells could reveal novel targets for the development of drugs to prevent oxidative damage and its subsequent pathological activation of downstream pathways deleterious to brain cell health. As such, drugs targeting these pathways may have antidepressant properties in humans, and could provide an alternative approach to treating depression and reducing suicide risk. In fact, we found that repeated exposure of rats to psychological stress increased DNA oxidation in prefrontal cortical white matter. Furthermore, preliminary findings using rat models of depression reveal that interruption of pathways downstream to oxidative damage produces a robust antidepressant response, correcting depressive-like behaviors elicited by psychological stress. These findings strongly implicate a role of oxidative damage in the etiology of MDD and possibly suicide, and demonstrate the utility of studying brain pathology as a logical path to identifying novel antidepressant targets.

Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.

Background: Pathology of white matter in brains of patients with major depressive disorder (MDD) is well-documented, but the cellular and molecular basis of this pathology are poorly understood. Methods:Levels of DNA oxidation and gene expression of DNA damage repair enzymes were measured in Brodmann area 10 (BA10) and/or amygdala (uncinate fasciculus) white matter tissue from brains of MDD (n=10) and psychiatrically normal control donors (n=13). DNA oxidation was also measured in BA10 white matter of schizophrenia donors (n=10) and in prefrontal cortical white matter from control rats (n=8) and rats with repeated stress-induced anhedonia (n=8). Results:DNA oxidation in BA10 white matter was robustly elevated in MDD as compared to control donors, with a smaller elevation occurring in schizophrenia donors. DNA oxidation levels in psychiatrically affected donors that died by suicide did not significantly differ from DNA oxidation levels in psychiatrically affected donors dying by other causes (non-suicide). Gene expression levels of two base excision repair enzymes, PARP1 and OGG1, were robustly elevated in oligodendrocytes laser captured from BA10 and amygdala white matter of MDD donors, with smaller but significant elevations of these gene expressions in astrocytes. In rats, repeated stress-induced anhedonia, as measured by a reduction in sucrose preference, was associated with increased DNA oxidation in white, but not gray, matter. Conclusions:Cellular residents of brain white matter demonstrate markers of oxidative damage in MDD. Medications that interfere with oxidative damage or pathways activated by oxidative damage have potential to improve treatment for MDD.

Multiple Sclerosis (MS) is a chronic, inflammatory disease of the central nervous system. MS is caused by the immune-mediated destruction of myelin and oligodendrocytes, resulting in demyelination and neurodegeneration. The microRNA miR-145-5p has been demonstrated to be upregulated in MS lesions. Our lab has previously shown that dysregulation of miR-145-5p can interfere with oligodendrocyte differentiation in mice and that knockout of miR-145-5p protects mice from experimental autoimmune encephalomyelitis (EAE), a model for MS. The objective of this study is to determine if inhibition of miR-145-5p with an antisense oligonucleotide (ASO) is sufficient to protect mice from EAE. Female mice were induced with EAE and then treated with a control or miR-145 ASO at the onset of disease. We evaluated disease progression by monitoring clinical severity, and evaluating molecular and structural characteristics of EAE by RT-qPCR, histology, immunohistochemistry and electron microscopy. We have shown that the miR-145 ASO reduced miR-145-5p expression in the lumbar spinal cord, spleen and thymus following EAE induction. Treatment with the miR-145-5p ASO resulted in improved clinical severity of EAE, reduced neuroinflammation and increased myelination. Inhibition of miR-145-5p may represent a novel treatment for MS.

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