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Mitogen-activated protein kinase kinase kinases (MAP3Ks) dual leucine kinase (DLK) and leucine zipper kinase (LZK) are essential mediators of axon damage responses, but their responses are varied, complex, and incompletely understood. To characterize their functions in axon injury, we generated zebrafish mutants of each gene, labeled motor neurons (MNs) and touch-sensing neurons in live zebrafish, precisely cut their axons with a laser, and assessed the ability of mutant axons to regenerate in larvae, before sex is apparent in zebrafish. DLK and LZK were required redundantly and cell autonomously for axon regeneration in MNs but not in larval Rohon-Beard (RB) or adult dorsal root ganglion (DRG) sensory neurons. Surprisingly, in dlk lzk double mutants, the spared branches of wounded RB axons grew excessively, suggesting that these kinases inhibit regenerative sprouting in damaged axons. Uninjured trigeminal sensory axons also grew excessively in mutants when neighboring neurons were ablated, indicating that these MAP3Ks are general inhibitors of sensory axon growth. These results demonstrate that zebrafish DLK and LZK promote diverse injury responses, depending on the neuronal cell identity and type of axonal injury.SIGNIFICANCE STATEMENT The MAP3Ks DLK and LZK are damage sensors that promote diverse outcomes to neuronal injury, including axon regeneration. Understanding their context-specific functions is a prerequisite to considering these kinases as therapeutic targets. To investigate DLK and LZK cell-type-specific functions, we created zebrafish mutants in each gene. Using mosaic cell labeling and precise laser injury we found that both proteins were required for axon regeneration in motor neurons but, unexpectedly, were not required for axon regeneration in Rohon-Beard or DRG sensory neurons and negatively regulated sprouting in the spared axons of touch-sensing neurons. These findings emphasize that animals have evolved distinct mechanisms to regulate injury si

The conserved MAP3K Dual-Leucine-Zipper Kinase (DLK) and Leucine-Zipper-bearing Kinase (LZK) can activate JNK via MKK4 or MKK7. These two MAP3Ks share similar biochemical activities and undergo auto-activation upon increased expression. Depending on cell-type and nature of insults DLK and LZK can induce pro-regenerative, pro-apoptotic or pro-degenerative responses, although the mechanistic basis of their action is not well understood. Here, we investigated these two MAP3Ks in cerebellar Purkinje cells using loss- and gain-of function mouse models. While loss of each or both kinases does not cause discernible defects in Purkinje cells, activating DLK causes rapid death and activating LZK leads to slow degeneration. Each kinase induces JNK activation and caspase-mediated apoptosis independent of each other. Significantly, deleting CELF2, which regulates alternative splicing of Map2k7, strongly attenuates Purkinje cell degeneration induced by LZK, but not DLK. Thus, controlling the activity levels of DLK and LZK is critical for neuronal survival and health.

The conserved MAP3K Dual-Leucine-Zipper Kinase (DLK) and Leucine-Zipper-bearing Kinase (LZK) can activate JNK via MKK4 or MKK7. These two MAP3Ks share similar biochemical activities and undergo auto-activation upon increased expression. Depending on cell-type and nature of insults DLK and LZK can induce pro-regenerative, pro-apoptotic or pro-degenerative responses, although the mechanistic basis of their action is not well understood. Here, we investigated these two MAP3Ks in cerebellar Purkinje cells using loss- and gain-of function mouse models. While loss of each or both kinases does not cause discernible defects in Purkinje cells, activating DLK causes rapid death and activating LZK leads to slow degeneration. Each kinase induces JNK activation and caspase-mediated apoptosis independent of each other. Significantly, deleting CELF2, which regulates alternative splicing of Map2k7, strongly attenuates Purkinje cell degeneration induced by LZK, but not DLK. Thus, controlling the activity levels of DLK and LZK is critical for neuronal survival and health.

La différenciation cellulaire est un processus complexe dont l’induction nécessite l’activation de certaines voies de signalisation. Dans le tissu nerveux, par exemple, il a été démontré que l’activation de la cascade de signalisation Dual Leucine Zipper Kinase (DLK) /Jun N-terminal Kinase (JNK) est essentielle pour la formation des neurites, ces prolongements du corps cellulaire des neurones qui servent de précurseurs aux dendrites et axones. L’un des effecteurs de cette voie, le facteur de transcription c-Jun, régule l’expression de plusieurs gènes dont ceux impliqués dans l’axonogenèse. Ce projet avait comme but principal de tester l’hypothèse que DLK contrôle une partie de l’axonogenèse dans les neurones en régulant l’expression des gènes clefs participants à ce processus. Pour y parvenir, la production de lignées clonales où Dlk est inactivée par le système CRISPR/Cas9 et la caractérisation de celles-ci a permis de valider que l’absence de DLK engendre un défaut de croissance des neurites dans les cellules Neuro-2a. Après qu’une analyse ChIP-seq de c-Jun ait été réalisé dans un modèle Neuro-2a différencié où DLK est déplétée, des gènes dont l’enrichissement de c-Jun à la chromatine variait en comparaison au contrôle ont été ciblés. Ensuite, l’effet de l’inactivation de DLK sur leur expression a été évalué par rt-qPCR. L’augmentation de l’expression des gènes Sémaphorine 6d (Sema6d) et Insulin like growth factor 1 receptor (Igf1r) a été constatée dans les lignées clonales en comparaison au contrôle. Cette hausse d’expression génique se traduit par l’augmentation du niveau protéique de SEMA6D. De plus, bien que le contrôle ne subisse aucune variation d’expression de Sema6d entre les conditions de prolifération et celles de différenciation, le niveau de SEMA6D atteint presque le triple pour les cellules lorsqu’elles se différencient. Les résultats de ce projet suggèrent que la voie DLK/JNK régule l’expression de ces gènes au niveau transcriptionnel de même que pos

Les rôles de DLK (‘dual leucine zipper kinase’) dans le système nerveux sont relativement bien décrits dans la littérature. Elle est, entre autres, impliquée dans la formation et la croissance des neurites. Cette propriété que possède DLK est attribuable, au moins en partie, à son effet sur le cytosquelette. Plusieurs protéines associées aux microtubules telles que DCX, Map1b et Map2 participent à l’assemblage des microtubules, l’élongation des neurites et sont parmi les effecteurs de l’axe signalisation qui médient la réorganisation cytosquelettique. Cependant, il existe plusieurs autres effecteurs de DLK et certains d’entre eux restent à être identifiés. Considérant l’importance de la régulation du cytosquelette dans la neuritogenèse et l’implication de DLK dans ce processus, on supposait que la recherche de ces effecteurs révèlerait des protéines fonctionnellement associées au cytosquelette ou en faisant partie. Nous désirions, donc, durant mon projet de maîtrise, contribuer à l’identification de ces effecteurs et caractériser le rôle particulier d’un de ces effecteurs dans la neuritogenèse. L’analyse comparative du phosphoprotéome des cellules Neuro-2a déplétées de DLK a révélé plusieurs peptides différentiellement phosphorylés dont deux phosphopeptides et, ainsi, deux sites de phosphorylation de la nestine (S1837, S894) étant significativement moins phosphorylés lorsque DLK est déplété. La génération de mutants et la quantification de leur expression ont permis d’observer la diminution de la stabilité de la nestine S894D/S1837D. De plus, la transfection du mutant S894A/S1837A a inhibé la croissance des neurites, ce qui suggère que la réduction de la stabilité de la nestine est nécessaire à la neuritogenèse. Elle est également fortement induite par la déplétion de la nestine. Ces observations suggèrent que DLK favorise la neuritogenèse en inhibant la nestine grâce à la régulation de sa phosphorylation aux résidus sérines 894 et 1837.

BackgroundTraumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown.MethodsHere we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI.ResultsOur findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival.ConclusionThe mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI.

BackgroundTraumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown.MethodsHere we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI.ResultsOur findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival.ConclusionThe mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI.

Damage to the central nervous system (CNS) such as from brain or spinal cord injury leads to permanent functional impairment, resulting in substantial social and economic burden. Sustained functional deficits are due in large part to the limitation of axon growth after injury. Damaged axons do not regenerate after CNS injury, whereas uninjured axons show some degree of spontaneous sprouting after CNS injury. Sprouting is believed to contribute to partial (albeit often insufficient) recovery in individuals with less severe injury or stroke, and can be promoted by manipulating axon growth regulators in model organisms, suggesting a potential approach to enhance recovery in humans. Nonetheless, our understanding of the molecular regulation of sprouting is incomplete. My dissertation work investigates the degree to which factors expressed by neurons and other CNS cell-types regulate axon sprouting and whether promotion of sprouting improves functional recovery from injury. I investigated the cell- type specific roles of a classically described myelin-associated inhibitor of axon growth, Nogo, and a novel neuron-intrinsic regulator of axon growth, DLK, in sprouting of the corticospinal tract (CST) after experimental unilateral pyramidotomy in mice. In order to evaluate whether promotion of CST sprouting correlates with improved functional recovery from injury, I evaluated and optimized behavioral tests to monitor functional recovery from pyramidotomy. I found that oligodendrocytic-specific deletion of Nogo enhances CST sprouting, showing for the first time that Nogo specifically expressed by oligodendrocytes restricts sprouting and spinal axon growth after CNS injury, supporting a longstanding but heretofore untested hypothesis. I was not able to detect a functional benefit associated with this enhanced sprouting, highlighting the challenge in harnessing structural plasticity to promote functional recovery. Additionally, I provide the first evidence for a role of DLK in

Damage to the central nervous system (CNS) such as from brain or spinal cord injury leads to permanent functional impairment, resulting in substantial social and economic burden. Sustained functional deficits are due in large part to the limitation of axon growth after injury. Damaged axons do not regenerate after CNS injury, whereas uninjured axons show some degree of spontaneous sprouting after CNS injury. Sprouting is believed to contribute to partial (albeit often insufficient) recovery in individuals with less severe injury or stroke, and can be promoted by manipulating axon growth regulators in model organisms, suggesting a potential approach to enhance recovery in humans. Nonetheless, our understanding of the molecular regulation of sprouting is incomplete. My dissertation work investigates the degree to which factors expressed by neurons and other CNS cell-types regulate axon sprouting and whether promotion of sprouting improves functional recovery from injury. I investigated the cell- type specific roles of a classically described myelin-associated inhibitor of axon growth, Nogo, and a novel neuron-intrinsic regulator of axon growth, DLK, in sprouting of the corticospinal tract (CST) after experimental unilateral pyramidotomy in mice. In order to evaluate whether promotion of CST sprouting correlates with improved functional recovery from injury, I evaluated and optimized behavioral tests to monitor functional recovery from pyramidotomy. I found that oligodendrocytic-specific deletion of Nogo enhances CST sprouting, showing for the first time that Nogo specifically expressed by oligodendrocytes restricts sprouting and spinal axon growth after CNS injury, supporting a longstanding but heretofore untested hypothesis. I was not able to detect a functional benefit associated with this enhanced sprouting, highlighting the challenge in harnessing structural plasticity to promote functional recovery. Additionally, I provide the first evidence for a role of DLK in

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord, for which there is no cure. Activation of the stress-induced c-Jun N-terminal kinase (JNK) signaling pathway, a critical mediator of neuronal apoptosis and axon degeneration, is evident in ALS and is therefore considered a potential target to prevent neurodegeneration. Pharmacological inhibition of an upstream activator of the JNK pathway, the mitogen-activated protein kinase kinase kinase (MAP3K12), has demonstrated neuroprotection in vitro as well as suppression of pathway activation in vivo. These observations, along with the largely neuronal-specific expression of MAP3K12, makes inhibition of this kinase a promising therapeutic target for ALS. Furthermore, a pharmacodynamic (PD) blood biomarker would be beneficial to help assess efficacy of MAP3K12 inhibition. A crucial step to interpreting the PD effects of MAP3K12 inhibition using blood biomarkers is to acquire evidence of MAP3K12 protein expression in blood, which was the aim of this study. First, we found Map3k12 transcript levels to be highly reduced in blood from Map3k12 conditional knockout (cKO) mice, demonstrating that these are excellent negative controls for studying MAP3K12 expression in blood. Next, three antibodies were identified that recognized mouse and human MAP3K12. Utilizing these antibodies, an intracellular flow cytometry method was developed that successfully detected MAP3K12 protein in human peripheral blood mononuclear cells (PBMCs). Collectively, our data provide evidence of MAP3K12 expression in human blood and thus pave the way for identifying PD blood biomarkers of MAP3K12 inhibition., Blood Biomarkers – a Window into the Brain? Little is known about the underlying cause of neurodegenerative diseases such amyotrophic lateral sclerosis (ALS). The protein MAP3K12 was recently identified as an important player in neurodegeneration and a potential therapeutic target for ALS. Our study found evidence of MAP3K12 in blood, which brings us closer towards identifying blood biomarkers to evaluate the biological effects of ALS drugs. ALS is a rare but terrifying neurodegenerative disease that kills half of the patients within 3 years from onset. The disease attacks the cells that are in charge of muscle movement, so called motor neurons. As the disease worsens, ALS patients lose their ability to move, speak, eat and ultimately breathe. After decades of research, there is still no cure to ALS. The cause of motor neuron death still puzzles scientists, but more clues are being revealed about the underlying disease mechanisms. Recent studies have uncovered a molecular pathway leading to degeneration of motor neurons in ALS. With increased attention to this pathway, a protein called MAP3K12 was found to be one of its key components. This new piece to the puzzle makes MAP3K12 a promising therapeutic target to prevent the death of motor neurons in people with ALS. These findings have led to development of drugs, so called inhibitors that block the activation of MAP3K12. However, the impossibility of collecting brain samples makes it difficult to measure the effect of these inhibitors within the brain. A way to overcome this obstacle is to use biomarkers. Biomarkers are measurable indicators that provide a window into health and disease. A classic example of a biomarker is the cholesterol levels in blood that serve as a risk indicator for heart disease. Another use of biomarkers is to assess the biological effects produced by a drug. Finding such biomarkers of MAP3K12 inhibitors would open up ways to monitor their effectiveness to help find the right dose for ALS pa

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

Le corps humain est composé de plusieurs milliards de cellules neuronales, lesquelles ont un impact primordial sur les systèmes vitaux. En effet, le système nerveux, étant lui-même un système vital du corps humain, effectue de nombreuses fonctions afin de voir au bon fonctionnement des autres systèmes du corps, tels que le système cardiovasculaire, le système digestif, le système musculaire et bien d’autres. Plusieurs maladies neurodégénératives telles que l’Alzheimer et la maladie de Creutzfeldt-Jakob, affectent les cellules nerveuses du corps et occasionnent la perte de différentes fonctions causant une diminution du bien-être et pouvant mener à la mort. La hausse du nombre d’individus atteint de maladies neurodégénératives observée au fils des ans nous montre l’importance de comprendre les phénomènes moléculaires qui se produisent lors des mécanismes de dégénérescence axonale dans les neurones. Lors de ma maîtrise dans le laboratoire du professeur Richard Blouin, j’ai étudié l’impact d’une augmentation intracellulaire de calcium sur la protéine dual leucine zipper kinase (DLK) en utilisant un modèle cellulaire humain, les SH-SY5Y. J’ai pu démontrer que l’entrée de calcium dans la cellule avait un impact sur la quantité de protéine DLK présente dans celles-ci. En effet, j’ai pu observer une diminution de la quantité de DLK dans les cellules suite à une entrée de calcium, un phénomène exclusivement calcium-dépendant. À l’aide d’inhibiteurs pharmacologiques, j’ai pu montrer l’implication, des calpaïnes, des protéases calcium-dépendantes reconnues pour leur rôle dans la dégénérescence axonale. Les essais in vitro montrent que DLK est une cible spécifique des calpaïnes.

Depuis plusieurs années, le tissu adipeux est reconnu comme un organe complexe et extrêmement important au maintien de l'homéostasie chez les mammifères. En effet, une dérégulation de sa taille et/ou de ses fonctions endocrines peut mener à l'apparition de nombreuses pathologies potentiellement mortelles, telles que le diabète de type II, l'hypertension ou encore des troubles cardiovasculaires. Ainsi, il est d'une importance capitale de bien comprendre les phénomènes moléculaires qui entourent la différenciation des cellules graisseuses afin de mieux réagir en clinique lors de l'apparition de telles conditions. Dans cette thèse, j'ai étudié le rôle d'une protéine kinase appelée dual leucine zipper-bearing kinase (DLK) dans la formation du tissu graisseux en utilisant un modèle cellulaire murin à différenciation inductible, les 3T3-L1. À l'aide d'approches pharmacologiques et d'ARN interférence, j'ai pu démontrer que cette protéine est essentielle à l'adipogenèse. En effet, une diminution de l'expression de DLK dans les cellules 3T3-L1 bloque la différenciation à un stade précoce, i.e. entre la liaison du facteur de transcription C/EBP[béta] à l'ADN et l'expression de ses gènes cibles, tels que C/EBP[alpha] et PPAR[gamma]. L'étude plus poussée des voies de signalisation des MAPKs à l'aide d'inhibiteurs pharmacologiques dans des cellules où l'expression de DLK a été diminuée a révélé que l'action bénéfique de DLK lors de la différenciation se situe au niveau de la voie ERK. En effet, l'induction de DLK au cours de l'adipogenèse résulte en une diminution de l'association des protéines KSR-1, Mek et ERK, ce qui mène à une diminution temporaire de l'activité de ERK et de la phosphorylation inhibitrice sur la sérine 82 du régulateur central de la différenciation des adipocytes, PPAR[gamma]. Finalement, nous avons pu déterminer que l'induction de DLK au cours de la différenciation des cellules graisseuses est modulée par PPAR[gamma].