Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1- class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1- mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days poststroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research. Aida Said Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Master of Science degree in Neuroscience Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa August 30, 2019 © Aida Said, Ottawa, Canada, 2019   Abstract Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1-class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1-mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days post-stroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research.

Indiana University-Purdue University Indianapolis (IUPUI) / The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19606号 / 医博第4113号 / 新制||医||1015(附属図書館) / 32642 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小池 薫, 教授 YOUSSEFIAN Shohab, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13213号 / 論医博第2163号 / 京都大学大学院医学研究科脳統御医科学系専攻 / (主査)教授 山下 潤, 教授 髙橋 良輔, 教授 木村 剛 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

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Sympathetic stimulation occurs in the heart after injuries such as ischemia/reperfusion (I/R) and myocardial infarction and affects myocardial remodeling. Prolonged sympathetic stimulation can result in myocardial dysfunction through its effects on cardiac myocyte apoptosis and myocardial fibrosis. Ubiquitin (UB) is well known for its role of tagging old or damaged proteins for degradation via the UB-proteosome pathway. The role of exogenous UB however, is not fully understood. Previously, our lab showed that β-adrenergic receptor (β-AR) stimulation increased levels of extracellular UB in the conditioned media of adult rat ventricular myocytes and that UB inhibits β-AR-stimulated apoptosis. This study investigates the role of extracellular UB after myocardial I/R injury in terms of infarct size, function, inflammation and proteomic changes in vivo as well as the effects of extracellular UB on cardiac fibroblast function in vitro. First, we validated a method of consistently measuring real-time myocardial ischemia and reperfusion in vivo. Second, cardiac function was studied 3 days post I/R injury in the presence or absence of UB infusion. Echocardiographic analysis determined UB infusion increased cardiac function after I/R injury in terms of ejection fraction and fractional shortening. UB decreased infarct size and infiltration of inflammatory cells including neutrophils and macrophages as well as reduced activity of neutrophils. UB increased protein levels of matrix metalloproteinase (MMP)-2 and transforming growth factor-β1 and increased activity of MMP-9. Third, in adult rat primary cardiac fibroblasts, we demonstrate that extracellular UB interacts with CXCR-4. UB treatment decreased serum-mediated increases in fibroblast proliferation and enhanced the contraction of fibroblast-populated collagen gels. Thus, extracellular UB likely interacts with CXCR-4 to influence fibroblast function and proliferation. Additionally, UB influences cardiac remodeling in terms of heart function, infarct size, inflammatory response and proteomic profile.

Ischemic heart disease represents a leading cause of death worldwide. Ischemia denotes an insufficient supply of oxygenated blood to the heart due to occlusion of the coronary vessels. Timely reperfusion, i.e., restoring blood flow to the ischemic part of the heart, limits ischemic damage. However, reperfusion itself induces injury to the heart. This phenomenon is referred as ischemia/reperfusion (I/R) injury. Osteopontin (OPN), also known as cytokine Eta-1, is a cell-secreted extracellular matrix protein. Expression of OPN increases in the heart in response to a variety of pathological conditions. Mice lacking OPN exhibit exaggerated left ventricular dilation in non-reperfused model of myocardial remodeling. Cardioprotective role of OPN has also been demonstrated in a mouse model of repetitive I/R injury for 7 days. The objective of this study was to examine the role of OPN in modulation of systolic and diastolic parameters of the heart following I/R injury in a time-dependent manner. For this study, wild type (WT) and OPN knockout (KO) mice aged ~4 months were subjected to cardiac ischemia by the ligation of left anterior descending coronary artery (LAD). Following 45 min of ischemia, the LAD was reperfused by snipping the ligature. Heart function was measured using echocardiography at baseline, 3, 7, 14, and 27 days following I/R injury. M-mode echocardiographic images were used to calculate the systolic parameters (% fractional shortening [%FS], % ejection fraction [%EF], and end-systolic volume [ESV]), while pulse wave Doppler images were used to calculate diastolic parameter (aortic ejection time; [AET]). Global cardiac function was evaluated using myocardial performance index (MPI; a Doppler-derived index which combines systolic and diastolic functions). At basal levels, most of the systolic and diastolic parameters remained unchanged between the two groups. I/R injury decreased %FS and EF in both groups vs the baseline values at 3, 7, 14 and 27 days post-I/R. However, the decrease in %FS and EF was significantly greater in KO-I/R vs WT-I/R group. ESV was significantly higher in WT mice 7 days post-I/R, and stayed higher 14 and 27 days post-I/R vs baseline. However, the increase in ESV was significantly greater in KO mice 3 day post-I/R, and remained higher vs WT-I/R during the time course. AET was lower in WT group 14 days post-I/R vs baseline. On the other hand, AET was significantly lower in KO group 3, 7, 14 and 27 days post-I/R vs WT-I/R. MPI was higher in WT group 7 days post-IR vs baseline. MPI decreased significantly in WT group 27 days vs 7 days post-I/R. In KO group, MPI was significantly higher than WT mice at baseline, and remained higher 3 and 27 day post-I/R vs WT-I/R. Thus, lack of OPN decreases systolic and diastolic functional parameters of the heart following I/R injury, suggesting a cardioprotective role of OPN in myocardial remodeling post-IR.