京都大学 / 新制・課程博士 / 博士(医学) / 甲第23070号 / 医博第4697号 / 新制||医||1049(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 花川 隆, 教授 溝脇 尚志, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Post-traumatic stress disorder (PTSD) is a heterogenous psychiatric condition that affects thousands of individuals each year. Of those who experience this condition, military members including members of the Canadian Armed Forces (CAF) are particularly vulnerable, demonstrating high prevalence rates of PTSD-related symptoms. Moreover, individuals with PTSD are at increased risk for comorbid conditions and are at greater risk for suicide due to the overwhelming, debilitating nature of PTSD symptoms. In previous research, hyperarousal symptoms associated with PTSD have been linked to dysregulation in the locus coeruleus norepinephrine (LC-NE) system, a vast neuromodulatory system responsible for regulating arousal, attention, autonomic and memory-related functions. Advancements in neuroimaging methods have advanced our ability to study connectivity in vivo such that small structures like the LC can be further studied in human samples. Specifically, neuromelanin-sensitive MRI (NM-MRI), a novel, non-invasive neuroimaging method has been shown to detect changes in neuromelanin (NM)-related signal in both the LC and substantia nigra (SN). NM is a dark pigment that accumulates over the lifespan in catecholamine-dominant centers such as the LC and SN and is the by-product of catecholamine oxidation. NM-MRI can be used to image these centers in vivo due to the paramagnetic properties offered by NM. Furthermore, when excess cytosolic catecholamine levels are present in select neurons, NM production is thought to be increased, resulting in increased NM signal from the LC. This could potentially be a marker for dysregulation as many conditions have been associated with variability of this system. Previously, NM-MRI has been used in other clinical settings such as in Parkinson’s disease (PD), Alzheimer’s disease (AD), schizophrenia and depression; however, this current investigation is the first to utilize this imaging modality in the context of PTSD. Specifically, we hypothesized that increased NM-MRI signal in the LC would correlate with increasing severity of hyperarousal symptoms in individuals with PTSD. We also predicted that the opposite would be true for comorbid depression symptom severity, as reduced LC signal has been previously correlated with clinical measures of comorbid depression using NM-MRI. As per our primary hypothesis, we observed a significant positive correlation between NM-MRI signals in the caudal elements of the LC with hyperarousal symptom severity in 22 PTSD subjects (r= 0.54, p= 0.017; partial correlation controlling for depression symptom severity, age, and sex). In contrast, we did not find any evidence to support our secondary hypothesis, because a non-significant trend correlating LC NM-MRI signal and depression symptom severity was obtained (r= -0.30, p=0.22; partial correlation controlling for hyperarousal severity, age, and sex). Based on these results, we were able to build on previously conducted work to further investigate the utility of NM-MRI in the detection of variability in LC-NE system as it pertains to psychiatric conditions known to show dysregulation of this system such as PTSD. In addition, this thesis provides further evidence to support the automation of NM-MRI analytical methods, thus supporting their potential utility for future clinical research. Our findings also provide support for the use of NM-MRI as a potential measure of NE activity; further, this work provided preliminary evidence supporting the use of NM-MRI in a clinical, psychiatric setting, where the technique may serve as a biomarker of PTSD pathology. With these findings in mind, additional validation studies can be conducted to verify the use of NM-MRI as a biomarker for NE system dysregulation. This would potentially allow for advancements in targeted treatment options for PTSD, particularly those targeting the LC-NE system, thus potentially increasing patient stratification and treatment efficacy.

Post-traumatic stress disorder (PTSD) is a psychiatric condition caused by exposure to a traumatic event. Veterans are at especially high risk of PTSD. In Canada, in a survey conducted in 2016, 16% of Regular Force Veterans released during 1998-2015 reported having PTSD, which is at least two times higher than the estimated prevalence rate in the general population. Individuals with PTSD experience a range of debilitating symptoms, such as the intrusion of unwanted and distressing memories, persistent flashbacks, hypervigilance and nightmares. The neuropathophysiological mechanisms underlying symptoms of PTSD are not well understood, which remains a significant barrier to developing effective treatments. Hallmark PTSD symptoms such as hyperarousal and sleep disturbances may be related to dysregulation of noradrenaline (NA), a neurotransmitter produced in the locus coeruleus (LC) known to modulate cognition, arousal and sleep. This thesis examines the possible associations between dysregulation in rapid-eye-movement (REM) sleep and dysfunction in NA-containing neurons in the LC among veterans with PTSD. Twenty-two operationally deployed veterans with a history of PTSD were recruited through the Royal Ottawa Mental Health Centre in Ontario, Canada. A novel, non-invasive neuroimaging method, neuromelanin-sensitive magnetic resonance imaging (NM-MRI), was used to detect a by-product of NA called neuromelanin (NM) in the LC of each participant. Then a contrast-to-noise ratio (CNR) was calculated to obtain a marker of the NA function. The LC was segmented into three subdivisions to assess whether the association between NM and REM sleep may differ across regions of the LC. As hypothesized, we observed different associations between NM and REM sleep across regions of the LC. After controlling for antidepressant usage, there was (i) a moderate, negative, significant correlation between the percentage of REM sleep and rostral LCCNR, r(19) = -.476, p = .029, (ii) a weak positive non-significant correlation between the percentage of REM sleep and caudal LCCNR, r(19) = .33, p = .145, and (iii) no significant correlation between REM sleep percentage and LCCNR in the middle LC, r(19) = -.04, p = .876. This thesis is the first study to show that NM and REM sleep may be related in veterans with PTSD and that this relationship may vary across subdivisions of the LC. These results improve understanding of REM sleep among individuals with PTSD. The results may stimulate the investigation of novel pharmacotherapy focused on sleep disturbances in PTSD, the development of personalized treatments for PTSD, and the search for clinical biomarkers of PTSD based on brain function. The current study also made methodological contributions that may be applicable beyond the research on PTSD to the field of REM sleep and the NA system. Specifically, the current study showed the suitability of the NM-MRI method for examining the connections between NM and REM sleep, and it showed that segmenting the LC can lead to a more nuanced understanding of its role in the human body.

Atualmente observa-se que a proporção de pessoas com 60 anos ou mais está crescendo mais rápido do que a de outras faixas etárias. Um dos resultados desta transição epidemiológica é o aumento das doenças cujo fator de risco é o envelhecimento, entre elas, a doença de Parkinson. Embora muitas regiões exibam os sinais neuropatológicos da doença de Parkinson, a degeneração dos neurônios, contendo neuromelanina, da substância negra é considerada como sendo uma característica importante, representando o critério cardinal para o diagnóstico. No entanto, ainda não está claro por que certas regiões do cérebro, como a substância negra, são vulneráveis em algumas doenças neurodegenerativas e alguns neurônios vizinhos, às vezes morfologicamente indistinguíveis, permanecem preservados. Análises moleculares de populações de neurônios podem conduzir a uma melhor compreensão sobre a fisiologia dos mesmos, bem como os mecanismos envolvidos nos processos de doença. Na era pós genômica, realizar análises proteômicas são de grande interesse científico, pois permitem avanços no conhecimento dos processos biológicos. A técnica de microdissecção e captura a laser tem sido uma ferramenta importante e cada vez mais utilizada para aquisição de populações puras de células a partir de secções histológicas, evitando que áreas não pertencentes ao tecido alvo sejam dissecadas. A união destes métodos pode contribuir de maneira relevante para o entendimento fisiopatológico dos neurônios contendo neuromelanina da substância negra. No entanto, para que a microdissecção e captura a laser e as análises proteômicas sejam eficazes, é imprescindível a aplicação de um protocolo bem estruturado. Dentro desse contexto, o presente trabalho tem como objetivo criar um protocolo de microdissecção a laser de neurônios contendo neuromelanina em indivíduos cognitivamente normais, para subsequente análise proteômica. Os casos utilizados neste estudo são provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. Para o desenvolvimento da nossa proposta, contamos com a colaboração do Centro de Proteômica Médica da Universidade de Bochum, Alemanha. O protocolo foi desenvolvido baseado em outros previamente descritos na literatura e otimizado de acordo com objetivos pretendidos. Analisamos o plano anatômico de amostragem do tecido, o método de congelamento, a espessura do corte para a microdissecção, a solução utilizada para a coleta do tecido durante a microdissecção e o método de digestão proteolítica para posteriores análises proteômicas. Através de ensaios comparativos, alcançamos os resultados desejados e os mesmos foram validados através de análises por espectrometria de massas. Consequentemente, também fomos capazes de reconhecer fatores técnicos que possivelmente impossibilitariam um efetivo estudo do proteoma / Currently the worldwide proportion of people aged 60 years and over is growing faster than any other age group. This strikingly epidemiological transition results in an increase of aging related diseases, including Parkinson\'s disease (PD). Although many brain areas exhibit the neuropathological signs of Parkinson\'s disease, the degeneration of neuromelanin containing cells in the substantia nigra is considered a hallmark feature, representing cardinal diagnostic criteria for PD. However, why certain brain regions -- such as the substantia nigra -- are vulnerable in some neurodegenerative diseases, while some neighboring morphologically indistinguishable neurons remain preserved, is still unclear. Molecular analysis of specific neuronal populations can lead us to a better understanding about the physiological role played by these neurons and mechanisms involved in disease\'s processes. In a post-genomic era, proteomic analyses are of great scientific interest since they allow a better understanding of the biological processes. The laser capture microdissection technique has also became an important tool in biological research, being increasingly used for acquisition of pure populations of cells from histological sections, preventing the dissection of areas outside the target tissue. The combination of these methods can significantly contribute to understand the pathophysiological role of the containing neuromelanin neurons of the substantia nigra. However, for an effective application of both techniques, laser capture microdissection and proteomic analysis, it is essential the application of an efficient protocol. In this context, this study aims to establish a protocol for laser microdissection of containing neuromelanin neurons in cognitively normal individuals for subsequent proteomic analyses. We selected cases from the Brain Bank of the Brazilian Aging Brain Study. A collaboration with the Medical Proteome Center, University of Bochum, Germany took part during the development of our proposal. Our protocol was developed based on previous published protocols and optimized according the intended aims. We analyzed anatomical planes for neuronal collection, freezing methods, thickness of tissue for microdissection sections, solution for tissue collection during laser microdissection and the proteolytic digestion methods. Through our comparative tests, we have achieved the desired results and validated them by mass spectrometry analyses. Consequently, we were also able to exclude technical factors that could possibly preclude one effective proteome analysis

Methamphetamine (METH) is a highly addictive substance that is highly prevalent in today’s society, with over 1 in 20 adults over 26 having taken it at least once. While it is known that METH, a common psychostimulant, acts on both the mesolimbic dopamine (DA) and nigrostriatal DA systems by affecting proteins involved in DA reuptake and vesicular packaging, the specific mechanism of what is known as METH neurotoxicity remains obscure, but has been shown to involve oxidative stress. Studies have shown that reactive oxygen species act on the same proteins that METH affects. Oxidative species have also been known to catalyze the formation of melanins in dopaminergic cells. We explore this link more fully here. In an in vitro system, oxidative species (including Fe3+, an inorganic catalyst for oxidative stress), enhance the rate of melanization of DA. Methamphetamine increased oxidative stress in an in vivo model. Additionally, METH enhanced phasic (stimulated) DA release and caused an electrically-independent efflux of DA. Lidocaine abolished phasic DA release, but did not affect METH-induced DA efflux, indicating action-potential dependent and independent mechanisms behind METH’s effects. The sigma-1 receptor antagonist BD 1063 significantly attenuated METH’s effect on DA release. Depletion of intracellular calcium (Ca2+) reserves also attenuated METH-enhancement of DA release. We investigated the role of oxidative species in METH-induced DA efflux. Reduced glutathione (the substrate for glutathione peroxidase) and 4-hydroxy-TEMPOL (a superoxide dismutase mimetic) blocked METH’s effect on DA release, suggesting that a reactive oxygen species (ROS), most likely superoxide, is necessary for METH-induced DA efflux. Finally, oxidative stress as well as acute METH impairs the vesicular monoamine transporter 2 (VMAT2) by S-glutathionylation modification of Cys-488, highlighting VMAT2 as a likely regulator of METH’s effects on electrically independent DA release. These findings help outline a model in which METH induces DA release in the NAc through a signaling cascade involving the sigma receptor and ROS signaling molecules.

Atualmente observa-se que a proporção de pessoas com 60 anos ou mais está crescendo mais rápido do que a de outras faixas etárias. Um dos resultados desta transição epidemiológica é o aumento das doenças cujo fator de risco é o envelhecimento, entre elas, a doença de Parkinson. Embora muitas regiões exibam os sinais neuropatológicos da doença de Parkinson, a degeneração dos neurônios, contendo neuromelanina, da substância negra é considerada como sendo uma característica importante, representando o critério cardinal para o diagnóstico. No entanto, ainda não está claro por que certas regiões do cérebro, como a substância negra, são vulneráveis em algumas doenças neurodegenerativas e alguns neurônios vizinhos, às vezes morfologicamente indistinguíveis, permanecem preservados. Análises moleculares de populações de neurônios podem conduzir a uma melhor compreensão sobre a fisiologia dos mesmos, bem como os mecanismos envolvidos nos processos de doença. Na era pós genômica, realizar análises proteômicas são de grande interesse científico, pois permitem avanços no conhecimento dos processos biológicos. A técnica de microdissecção e captura a laser tem sido uma ferramenta importante e cada vez mais utilizada para aquisição de populações puras de células a partir de secções histológicas, evitando que áreas não pertencentes ao tecido alvo sejam dissecadas. A união destes métodos pode contribuir de maneira relevante para o entendimento fisiopatológico dos neurônios contendo neuromelanina da substância negra. No entanto, para que a microdissecção e captura a laser e as análises proteômicas sejam eficazes, é imprescindível a aplicação de um protocolo bem estruturado. Dentro desse contexto, o presente trabalho tem como objetivo criar um protocolo de microdissecção a laser de neurônios contendo neuromelanina em indivíduos cognitivamente normais, para subsequente análise proteômica. Os casos utilizados neste estudo são provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. Para o desenvolvimento da nossa proposta, contamos com a colaboração do Centro de Proteômica Médica da Universidade de Bochum, Alemanha. O protocolo foi desenvolvido baseado em outros previamente descritos na literatura e otimizado de acordo com objetivos pretendidos. Analisamos o plano anatômico de amostragem do tecido, o método de congelamento, a espessura do corte para a microdissecção, a solução utilizada para a coleta do tecido durante a microdissecção e o método de digestão proteolítica para posteriores análises proteômicas. Através de ensaios comparativos, alcançamos os resultados desejados e os mesmos foram validados através de análises por espectrometria de massas. Consequentemente, também fomos capazes de reconhecer fatores técnicos que possivelmente impossibilitariam um efetivo estudo do proteoma / Currently the worldwide proportion of people aged 60 years and over is growing faster than any other age group. This strikingly epidemiological transition results in an increase of aging related diseases, including Parkinson\'s disease (PD). Although many brain areas exhibit the neuropathological signs of Parkinson\'s disease, the degeneration of neuromelanin containing cells in the substantia nigra is considered a hallmark feature, representing cardinal diagnostic criteria for PD. However, why certain brain regions -- such as the substantia nigra -- are vulnerable in some neurodegenerative diseases, while some neighboring morphologically indistinguishable neurons remain preserved, is still unclear. Molecular analysis of specific neuronal populations can lead us to a better understanding about the physiological role played by these neurons and mechanisms involved in disease\'s processes. In a post-genomic era, proteomic analyses are of great scientific interest since they allow a better understanding of the biological processes. The laser capture microdissection technique has also became an important tool in biological research, being increasingly used for acquisition of pure populations of cells from histological sections, preventing the dissection of areas outside the target tissue. The combination of these methods can significantly contribute to understand the pathophysiological role of the containing neuromelanin neurons of the substantia nigra. However, for an effective application of both techniques, laser capture microdissection and proteomic analysis, it is essential the application of an efficient protocol. In this context, this study aims to establish a protocol for laser microdissection of containing neuromelanin neurons in cognitively normal individuals for subsequent proteomic analyses. We selected cases from the Brain Bank of the Brazilian Aging Brain Study. A collaboration with the Medical Proteome Center, University of Bochum, Germany took part during the development of our proposal. Our protocol was developed based on previous published protocols and optimized according the intended aims. We analyzed anatomical planes for neuronal collection, freezing methods, thickness of tissue for microdissection sections, solution for tissue collection during laser microdissection and the proteolytic digestion methods. Through our comparative tests, we have achieved the desired results and validated them by mass spectrometry analyses. Consequently, we were also able to exclude technical factors that could possibly preclude one effective proteome analysis

RCSN-3 cells are a cloned cell line derived from the substantia nigra of an adult rat. The cell line grows in monolayer and does not require differentiation to express catecholaminergic traits, such as (i) tyrosine hydroxylase; (ii) dopamine release; (iii) dopamine transport; (iv) norepinephrine transport; (v) monoamine oxidase (MAO)-A expression, but not MAO-B; (vi) formation of neuromelanin; (vii) vesicular monoamine transporter-2 (VMAT-2) expression. In addition, this cell line expresses serotonin transporters, divalent metal transporter, DMT1, dopamine receptor 1 mRNA under proliferating conditions, and dopamine receptor 5 mRNA after incubation with dopamine or dicoumarol. Expression of dopamine receptors D2, D3 and D4 mRNA were not detected in proliferating cells or when the cells were treated with dopamine, CuSO4, dicoumarol or dopamine-copper complex. Angiotensin II receptor mRNA was also found to be expressed, but it underwent down regulation in the presence of aminochrome. Total quinone reductase activity corresponded 94% to DT-diaphorase. The cells also express antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. This cell line is a suitable in vitro model for studies of dopamine metabolism, since under proliferating conditions the cells express all the pertinent markers.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24786号 / 医博第4978号 / 新制||医||1066(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 花川 隆, 教授 村井 俊哉, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. </p><p>In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis.</p><p>The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[<i>a</i>]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. </p><p>These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.</p>

The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis. The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.