Les douleurs neuropathiques ayant une origine à la suite de blessures traumatiques du SNC ou du SNP sont particulièrement difficiles à traiter en utilisant les moyens thérapeutiques actuellement disponibles. Il est donc nécessaire d'identifier de nouvelles stratégies thérapeutiques. Notre objectif était donc de définir les mécanismes impliqués dans ces douleurs neuropathiques. LIMK1 est l'un des acteurs possibles de la réorganisation épinière qui caractérise les lésions nerveuses. Une fonction très caractérisé de cette protéine, est la phosphorylation d'une famille de protéines appelées « cofilines ». Sa phosphorylation, ce qui induit la réorganisation du cytosquelette d'actine. Récemment, il a été montré qu’un microARN (miARNs) nomé miR-134 régule l'expression de LIMK1 en se liant au messager de LIMK1 (ARNm), inhibant sa traduction en protéine physiologiquement active. Notre hypothèse était que la régulation de LIMK1 par miR-134 pourrait jouer un rôle essentiel dans la sensibilisation à la douleur. Cette régulation pourrait ainsi être liée non seulement à la modulation neurochimique neuronale mais aussi à la plasticité fonctionnelle associée. Au cours de cette thèse, l’HIS a montré une diminution de miR-134 chez des rats SNL (neuropathique), cette sous-expression était concomitante à une augmentation de LIMK1 illustrée par l’IHC. Il est important de noter ici que l'ISH est une méthode de détection connue récemment et qui a été identifiée pour visualiser les miARNs. Des différents protocoles de l’HIS ont également été discutés dans le cadre de cette thèse. Ce résultat a été confirmé par Le qRT-PCR . Par la suite, afin de vérifier les changements comportementaux douloureux induits par miR-134 et LIMK1. Nous avons effectués des injections intrathécales de siRNA anti-LIMK1 pour inhiber l'expression endogène de LIMK1 chez les SNL. C’était intéressant de ne pas avoir trouvé aucun changement comportemenal chez les SNL après ce type d’injection. Une surexpression artificielle de miR-134 en utilisant un précurseur de miR-134 (premiR-134) chez les SNL a montré le même effet. Ensuite, nous avons essayé d'effectuer les mêmes injections chez les Sham (control), et c’était plus intéressant de trouver que ces injections (siRNA LIMK1 et premiR-134) ont provoqué une hypersensibilité douleureuse chez les sham. Cela a été illustré au moyen de deux tests de comportement; le Von Frey (VF) et la distribution pondérale dynamique (DWB). Pour etudier l'effet inverse, nous avons inhibé miR-134 en utilisant une sonde spécifique KD (Knock-Down); une diminution significative inattendue dans le seuil de retrait a été observée avec VF et DWB. qRT-PCR dans la plupart de ces cas, a confirmé la corrélation in vivo entre miR-134 et LIMK1. Enfin, nous avons cherché un mécanisme d'action possible qui pourrait réguler cette modulation. Des données récentes publiées ont montré une implication de l'ADF/cofiline sur le trafic des récepteurs AMPA (AMPAR). En accord avec les résultats mentionnés ci-dessus, la transfection du KD de miR-134 a montré une diminution dans AMPAR adressés à la membrane plasmique. Tout ensemble ces données suggèrent que l'effet antinociceptif de KD de miR-134 et la surexpression de LIMK1 sont indirectement régulé par l'insertion des AMPAR à la membrane plasmique.Il semble que miR-134 exerce un effet différent sur la douleur neuropathique que miR-103, discuté aussi dans le cadre de cette thèse. Il était demontré comme un régulateur de plusieurs cibles, les trois sous-unités formant les canaux calciques de type-L « Cav1.2 LTC ». MiR-103 a été trouvé également réprimés chez les SNL. La surexpression de miR-103 soulage la douleur neuropathique. Contrairement au miR-134, miR-103 exerce un rôle pronociceptive pendant la douleur neuropathique. / Pains having a neuropathic origin following CNS or PNS traumatic injury are particularly difficult to treat using the actually available therapeutic means. It is thus necessary to identify new therapeutic strategies. Hence, our aim was to define the mechanisms implicated in these neuropathic pains. Nervous lesions are characterised by an anatomical reorganization of the neuronal network of the dorsal horn. Neurochemical alterations are also involved. Some of the molecular mechanisms underlying the neuronal plasticity (a main feature of neuropathic pain) have been emphasized here by a variety of complementary technical approaches. LIMK1 is one of the possible actors of this reorganization. Among this protein’s known functions, and the most characterized is the phosphorylation of a family of proteins known as cofilins. Their phosphorylation induces the reorganization of actin cytoskeleton. Recently, it has been shown that a miR-134 miRNA regulates LIMK1 expression by binding to the LIMK1 messenger, inhibiting its translation into physiologically active protein. Our hypothesis is that LIMK1 regulation by miR-134 might play an essential role in pain sensitization by modulating neuron neurochemical reorganization and the associated functional neuronal plasticity. Firstly, by means of IHC and ISH, we studied miR-134/LIMK1 distribution within the dorsal horn of the spinal cord in sham animal (control group) and in neuropathic pain model (SNL model). Important to note here that ISH is a known detection method recently identified to visualize miRNA. Different protocols of ISH were discussed in a part of this thesis. ISH showed a decrease in miR-134 expression in SNL rats concomitantly with an increase in LIMK1 illustrated by IHC. This finding has been confirmed by qRT-PCR techniques. Afterward, in order to check for the possible behavioural-induced changes of miR-134 and LIMK1. We intrathecally injected an anti-LIMK1 siRNA to inhibit endogenous LIMK1 expression in SNL rats. Interestingly no significant changes in pain behaviour have been observed. Artificial overexpression of miR-134 using a PremiR-134, showed the same effect. Then we tried to perform the same injections on sham rats, and more interestingly, siRNA LIMK1 and premiR-134 evoked pain hypersensitivity in shams rats. This was illustrated by means of two behaviour tests; Von Frey (VF) and the Dynamic Weight bearing (DWB). To explore the reverse effect, we inhibited miR-134 using a specific KD probe in SNL rats; unexpectedly a significant decrease in pain withdrawal threshold was observed with VF and DWB. qRT-PCR in most cases confirmed the in vivo correlation between miR-134 and LIMK1. Finally, we searched for the possible mechanism of action that could regulate this modulation. Recent published data showed an involvement of ADF/cofilin on AMPAR trafficking. In line with the above mentioned findings, miR-134 KD transfection showed a decrease in AMPAR addressed to the plasma membrane. Altogether suggest that the antinociceptive effect of miR-134 KD and LIMK1 overexpression are mediated by AMPAR insertion at the plasma membrane. It seems that miR-134 exerts a different effect on neuropathic pain than miR-103 another miRNA discussed within the frame of this thesis. MiR-103 has been proved to regulate multiple targets, the three subunits forming Cav1.2 LTC. Pain sensitization involves Cav1.2 activation which consequently alters gene expression during this form of plasticity. MiR-103 was found downregulated also in the SNL model. Conversely to miR-134, overexpression of miR-103 partially alleviates pain. It decreases pain withdrawal threshold of the Von Frey test. Unlike miR-134, miR-103 exerts a pronociceptive role during neuropathic pain.

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

LIM Kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, has been shown to be involved in cell cycle progression. In this study we examine the role of LIMK1 in G1 phase and mitosis. We found ectopic expression of LIMK1 resulted in altered expression of p27Kip1, the G1 phase Cyclin D1/Cdk4 inhibitor. Overexpression of LIMK1 resulted in lower levels of p27Kip1 and p27Kip1-pY88 (inactive p27Kip1). Knockdown of LIMK1 resulted in elevated levels of p27Kip1 and p27Kip1-pY88. Together, these results suggest LIMK1 regulates progression of G1 phase through modulation of p27Kip1 expression. LIMK1 is involved in the mitotic process through inactivating phosphorylation of Cofilin. Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with ?-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one other's function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation. The substrates of LIMK1, Aur-A and Cofilin, are also involved in the mitotic process. Aur-A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through regulation of the actin cytoskeleton. Aur-A phosphorylates Cofilin at multiple sites including S3 resulting in inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during progression of prophase to metaphase. Inhibition of Aur-A activity altered subcellular localization of Cofilin and induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the early mitotic stage through regulation of actin cytoskeleton reorganization. ?

LIM kinase 1 (LIMK1) is a unique dual specificity serine/threonine kinase containing two N-terminal LIM domains in tandem, a PDZ domain and a C-terminal catalytic domain. LIMK1 is involved in modulation of actin cytoskeleton through inactivating phosphorylation of the ADF (actin depolymerization factor) family protein cofilin. Recent studies have shown that LIMK1 is upregulated in breast and prostate cancer cells and tissues, promotes metastasis in animals and induces acquisition of an invasive phenotype when ectopically expressed in benign prostate epithelial (BPH) cells. Furthermore, overexpression of LIMK1 was associated with altered sub cellular localization of the membrane type 1 matrix metalloprotease (MT1-MMP). Matrix metalloproteases (MMPs) are a family of zinc dependant proteolytic enzymes that hydrolyze extra cellular matrix and cell surface molecules. A number of MMPs including MMP-2, MMP-9 and their activator MT1-MMP are over expressed in a variety of cancers including prostate cancer. The abundant expression of these enzymes contributes to changes in the tumor microenvironment, which facilitate degradation of the surrounding collagen matrix and migration of cells through the matrix defects. In this study, we show that MMPs are involved in LIMK1 induced invasion of otherwise non-invasive BPH cells. We also show that (a) the kinase activity of LIMK is not essential for the invasive behavior of the cells and (b) the absence of LIM domains significantly retards cell invasion. We have established transfected sub lines of BPH cells stably expressing 1) constitutively active LIMK1 (BPHLCA), 2) kinase dead LIMK1 (BPHLKD) and 3) only the kinase domain of LIMK1 (BPHLK) for our study. In vitro invasion assays revealed that LIMK1 induced invasion was inhibited by the MMP specific inhibitor, GM6001, and that cells expressing kinase-dead LIMK1 were equally invasive. Furthermore, BPH cells expressing LIMK1 mutants expressed higher amounts of MMP-2 and MMP-9. Substrate zymography revealed increased concentration of secreted MMP-2 and MMP-9 in the media of BPHLCA and BPHLK cells respectively compared to BPHV (vector control) cells. Quantitative RT-PCR also showed a ~10 fold increase in the steady state concentration of MMP-2 in BPHLCA cells compared to the control BPHLV cells. Expression of active LIMK1 stimulated cell-surface expression of MT1-MMP in BPHLCA cells as determined by flow cytometry. A modest increase in expression of MT1-MMP was noted in BPHLKD cells compared to BPHLK and BPHV cells. Immunoflourescence analysis indicated differential localization of MT1-MMP and LIMK1 in BPH cells expressing different mutants of LIMK1. Co-localization of LIMK1 and MT1-MMP in the plasma membrane and in the perinuclear region was also evident in these cells. Furthermore, here we provide evidence that suggests a functional role for phosphorylated (activated) LIMK1/2 (p-LIMK1/2) during mitosis through its association with γ-tubulin. Immunoflourescence analysis showed distinct co-localization of γ -tubulin and p-LIMK1/2 in the centrosomes during mitosis from early prophase to the beginning of telophase. No association was seen in the interphase or in late telophase. Phospho-LIMK1/2 was co-precipitated in immunoprecipitates of γ -tubulin using an anti- γ -tubulin antibody suggesting a physical association between these proteins in a complex. This finding reveals a novel role of LIMK1 in the mitotic process. In summary, our data suggests that MMPs are involved in LIMK1 induced invasion of prostate epithelial cells, and that this effect is mediated through altered expression and activation of specific MMPs. Furthermore, LIMK1 induced invasion is dependant on the presence of LIM domains more than the kinase activity. Finally, we show that phosphorylated LIMK1 and LIMK2 are involved in the mitotic process in a stage specific manner through its association with the centrosomal protein γ -tubulin. Because LIMK1 promotes invasion in vitro, regulates expression of MMPs, and is involved in mitotic processes, it is an attractive drug target for prostate cancer therapy.