Class of 2014 Abstract / Specific Aims: The specific aim of this study was to measure the severity of dopamine receptor 1 (D1R)- and dopamine receptor 2 (D2R)-induced abnormal involuntary movements (AIMs) when administered with the NMDA receptor antagonist MK-801 or opioid glycopeptide MMP-2200. Methods: Male Sprague-Dawley rats were injected with 6-hydroxydopamine to cause unilateral loss of dopaminergic neurons in the substantia nigra and subsequent striatal dopamine loss. Levodopa (7 mg/kg; i.p.) injection for 21 consecutive days caused the rats to develop levodopa-induced dyskinesias, termed AIMs in this preclinical rat model. The rats were first primed with the D1R agonist SKF81297, then co-administered with MK-801 or MMP-2200 and AIMs scores were recorded to determine the severity of the dyskinesias. Then the same procedure was performed with the D2R agonist quinpirole. Main Results: MK-801 worsened D1R-induced limb, axial and orolingual (LAO) AIMs (p<0.05) whereas there was no change in locomotor AIM scores. MK-801 reduced D2R-induced LAO AIMs by 89% (p<0.001). However, MK-801 induced ipsiversive rotations, which is a parkinsonian symptom in this model. MMP-2200 had no effect on D1R-induced LAO AIMs but significantly reduced locomotor AIMs by 50% (p<0.05). MMP-2200 significantly decreased both D2R-induced LAO and locomotor AIMs by 40% and 90%, respectively (p<0.01). Conclusion: Both MK-801 and MMP-2200 had differential effects on the rodent direct and indirect striatofugal pathways with regards to AIMs. These results support that MK-801, an NMDA receptor antagonist, and MMP-2200, a mixed mu and delta opioid receptor agonist, modulate levodopa-induced dyskinesias through the dopaminergic and glutaminergic pathways.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

La dissémination des cellules cancéreuses et la formation de métastases sont des étapes cruciales dans la progression tumorale et constituent une cause majeure des décès dus au cancer. La métalloprotéase transmembranaire MT1-MMP est un acteur clé impliqué dans le franchissement des barrières tissulaires et le remodelage de la matrice extracellulaire (ECM) par les cellules cancéreuses. MT1-MMP est présente dans des vésicules intracellulaires, appelées endosomes, via lesquels elle est adressée à la membrane plasmique (PM) afin d'y dégrader la ECM. Des travaux menés au laboratoire ont identifié le complexe exocyste (CE) comme un acteur important pour la formation d'invadopodes dans la lignée d'adénocarcinome mammaire MDA-MB-231. Ce complexe multiprotéique (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 et Exo84) est impliqué dans l'arrimage des vésicules intracellulaires à la PM. Des cribles double-hybride ont identifiés la protéine WASH comme partenaire potentiel du CE (via les sous-unités Exo84 et Sec3). WASH est capable d'induire la polymérisation de l'actine en activant le complexe Arp2/3. In vitro, nous avons montré que les complexes WASH et exocyste interagissent physiquement et coordonnent le trafic intracellulaire et l'adressage de MT1-MMP à la PM. Ces résultats mettent en évidence une étroite collaboration entre le cytosquelette d'actine et les mécanismes d'exocytose lors des étapes précoces de dégradation de la ECM ainsi que dans l'invasion tumorale. / Cancer cell invasion is a prerequisite to tumor progression and metastasis. In order to disseminate, tumor cells must degrade and remodel the extracellular matrix (ECM) in a process that requires the trans-membrane matrix metalloproteinase MT1-MMP, which is a key component of the ECM remodeling apparatus of cancer cells. MT1-MMP overexpression in cancers is associated with increased invasion and metastasis. Many cellular proteins are involved in the transport and delivery of MT1-MMP-containing vesicles to the PM. Previous work from the laboratory identified the exocyst complex (EC) as a key component required for matrix proteolysis and invasion of cancer cells. This multiprotein complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) plays essential roles in docking secretory vesicles at the PM for exocytosis. To better characterize this complex, a yeast two-hybrid screen was performed, identifying the protein WASH as a potential partner of Exo84 and Sec3. WASH is a Nucleation Promoting Factor (NPF) able to activate the actin nucleating Arp2/3 complex. Results of the present study showed that WASH and the exocyst complexes interact and localize on MT1-MMP-positive endosomes in MDA-MB-231 breast cancer cells. This study highlight a direct implication of WASH and exocyst complex in ECM degradation by cancer cells through the docking and exocytosis of MT1-MMP-containing endosomes at the PM through connections between these compartments and the extracellular medium. This WASH- and exocyst-dependent MT1-MMP exocytosis mechanism is required for degradation of adjacent tissue by cancer cells during tumour cell invasion.

Matrix metalloproteinases (MMPs), a family of extracellular endopeptidases, are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Interleukin-1β (IL-1β), increased in the heart post-myocardial infarction (post-MI), plays a protective role in the pathophysiology of left ventricular (LV) remodeling following MI. Here we studied expression of various angiogenic genes affected by IL-1β in cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of MMP-2. cDNA array analysis of 96 angiogenesis-related genes indicated that IL-1β modulates the expression of numerous genes, notably increasing the expression of MMP-2, not MMP-9. RT-PCR and Western blot analyses confirmed increased expression of MMP-2 in response to IL-1β. Gelatin in-gel zymography and Biotrak activity assay demonstrated that IL-1β increases MMP-2 activity in the conditioned media. IL-1β activated ERK1/2, JNKs, and protein kinase C (PKC), specifically PKCα/β1, and inhibition of these cascades partially inhibited IL-1β-stimulated increases in MMP-2. Inhibition of PKCα/β1 failed to inhibit ERK1/2. However, concurrent inhibition of PKCα/β1 and ERK1/2 almost completely inhibited IL-1β-mediated increases in MMP-2 expression. Inhibition of p38 kinase and nuclear factor-κB (NF-κB) had no effect. Pretreatment with superoxide dismutase (SOD) mimetic, MnTMPyP, increased MMP-2 protein levels, whereas pretreatment with SOD and catalase mimetic, EUK134, partially inhibited IL-1β-stimulated increases in MMP-2 protein levels. Exogenous H2O2 significantly increased MMP-2 protein levels, whereas superoxide generation by xanthine/xanthine oxidase had no effect. This in vitro study suggests that IL-1β modulates expression and activity of MMP-2 in CMECs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20221号 / 医博第4180号 / 新制||医||1019(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 椛島 健治, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Matrix metalloproteinases (MMPs) are known to be the mediators of extracellular matrix remodeling. Increased levels of matrix metalloproteinases, particularly MMP-9, have been found in the aqueous humor of patients with glaucoma. However the exact role of MMP-9 in glaucomatous changes is not understood. Previous results from the West-Mays’ lab indicated that MMP-9 deficient (knockout - KO) mice exhibit elevated IOP, in the absence of distinct morphological changes in the anterior chamber. In the current thesis, I investigated whether the elevated IOP in MMP-9KO mice leads to RGC death. Wild type and KO littermates at different age groups: 2-3 months, 3-4 months, 6-8 and 9-12 months were studied. IOP was measured using TonoLab rebound tonometer. My results demonstrated that IOP was significantly increased in MMP-9KO mice compared to control littermates at all ages examined. To investigate if the elevated IOP was due to a difference in central corneal thickness (CCT), CCT measurements were made between WT and KO mice using ultrasound pachymeter. There was no difference in CCT demonstrating that the elevated IOP observed in MMP-9KO mice was not related to changes in corneal thickness. To determine whether the elevated IOP led to RGC death, the animals were sacrificed, eyes were enucleated and retinas (n=4) from both WT and KO animals were dissected and stained with Brn-3a antibody. Additional eyes were harvested from both WT and KO mice for histological and immunofluorescence studies. I found no observable difference in Brn3a+ RGC count between MMP9-WT and KO mice. Furthermore, no difference in retinal morphology, glial reactivity and laminin expression between WT and KO mice was observed. In the future it will be important to investigate whether elevated IOP in the MMP-9KO mice leads to optic nerve axonal loss and further investigate the possibility that the MMP-9KO retina is neuroprotected. / Thesis / Master of Science (MSc)

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Spinal cord injury (SCI) often leads to irreversible damage, and permanent paralysis inferior to the injury is common (Leibinger et al., 2013). Injury to the spinal cord occurs in two phases. In the first phase, components of the spinal cord are subject to mechanical trauma causing direct damage. In the second phase, damage spreads from the area of injury through molecular processes. Several studies have linked M1 "pro-inflammatory" macrophages to exacerbation of damage by inducing dieback of dystrophic axons, but not healthy axons, through direct cellular contact. Several studies have identified the presence of macrophage subtypes at specific time. A literature review was conducted in order to summarize these findings (Busch, Horn, Silver, & Silver, 2009; Evans et al., 2014; Horn, Busch, Hawthorne, van Rooijen, & Silver, 2008; Kigerl et al., 2009; Shechter et al., 2013). Although the full mechanism behind the process of M1 macrophage-mediated dieback of dystrophic axons is unclear, matrix metalloproteinase-9 (MMP-9) produced by these macrophages has been shown to play a role. However, the specific interaction between MMP-9 and neurons is under investigation. The research described explores the relationship between MMP-9 and fractalkine (CX3CL1), a surface protein expressed by CNS neurons. SDS-PAGE and western blot were used to determine whether the presence of MMP-9 increases the cleavage of fractalkine at several time intervals. At a concentration of 300ng/ml, MMP-9 was not found to demonstrate cleavage of fractalkine.

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