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Usp9x regulates axon specification and growth
Abstracts / Int. J. Devl Neuroscience 47 (2015) 1–131
increased in the group injected with inhibitor of ERK1/2 , Akt or EGF receptor. In conclusion, Fn has deteriorating effect on the recovery of SCI, which was not only dependent on itself activity, but also attributed to increase the expression of P2Y1 receptor via which ATP caused more astrocytic proliferation and inflammatory response. http://dx.doi.org/10.1016/j.ijdevneu.2015.04.064 ISDN2014 0079 Deubiquitylating enzyme Usp9x regulates neural progenitor fate in vivo Susitha Premarathne ∗ , Mariyam Murtaza, Stephen Wood Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia Neural progenitors (NP) give rise to all the cell types in the brain. During development NPs must balance self-renewal, and hence maintain a stem cell pool, with differentiation into neuron and glia. Regulation of NP fate occurs at multiple levels including post-translational protein modifications by ubiquitylation. Despite the fact that an ubiquitin pathway regulates most aspects of cell function, very little is known of its role in NPs. This is especially true for deubiquitylating enzymes, which remove ubiquitin from proteins and protect them from degradation. Usp9x is a deubiquitylating enzyme and a putative stemness gene highly expressed in NPs. Ectopic expression of Usp9x in embryonic stem cell derived NPs, promotes their self-renewal and polarity. Conversely, conditional in-vivo deletion of Usp9x from mouse NPs resulted in perinatal lethality and disorganisation of the neocortex and allocortex (Stegeman, 2013). Histological and immunoblot analyses of Usp9x-null NPs revealed decreased expression of several definitive adhesion and polarity marker proteins at E12.5. However, most of these markers recovered to normal levels by E14.5. In contrast, the adherens junctional and Wnt signalling molecule, -catenin, showed an increased expression level at both stages. Transcriptomic analysis revealed that the Wnt signalling pathway was significantly increased in the Usp9x-null telencephalon at E14.5. Interestingly, increased -catenin levels were detected in knockout NPs with both decreased (E12.5) and normal (E14.5) levels of N-cadherin suggesting this is an adhesion independent regulatory mechanism. Examination of NP fate at E12.5 revealed a premature differentiation of apical progenitors into abventricularly proliferating Tbr2 positive intermediate NPs. This study for the first time identifies deubiquitylating enzyme mediated post-translational modifications as a novel mechanism of NP fate determination in-vivo.
ISDN2014 0080 Usp9x regulates axon specification and growth M. Tan 1,∗ , L.A. Jolly 2 , M. Murtaza 1 , J. Camonis 3 , S.A. Wood 1 1 Eskitis Institute, Griffith University, Brisbane, Australia 2 SA Pathology, Women’s and Children’s Hospital, North Adelaide, Australia 3 Institute Curie, Paris, France E-mail address: men.tan@griffithuni.edu.au (M. Tan).
Establishing axon-dendrite polarity is essential for unidirectional signal transmission and the formation of complex neural circuits. Defective axon formation can lead to neurodegenerative and neurocognitive diseases. Conditional deletion of the deubiquitylating enzyme, Usp9X results in shorter axons in hippocampal neurons at 3, 5 and 7 days in culture. To study the role of Usp9X in axonal specification we compared wild type and Usp9X-null hippocampal neurons in the first 48 h of culture and categorised them into three stages. Stage 1 neurons extend a prominent lamella around the cell body. Stage 2 neurons extend short symmetrical neurites. During the transition from stages 2 to 3, neurite symmetry is broken and one neurite is specified to become an axon. From 12 h onward Usp9X-null neurons significantly (p = 0.002) lagged behind wild-type in progression from stages 1 to 2. However despite a lag of about 10 h the overall kinetics for stages 1 to 2 progression was similar. A more dramatic effect of Usp9X loss was during stages 2 to 3 progression where Usp9X-null neurons required over 36 h longer to specify an axon (p = 0.001). After this lag Usp9X-null neurons entered stage 3 at a similar rate to wild-type. Therefore Usp9X is required for axon specification. Using yeast-2-hybrid screening we identified an interaction between the kinesin1 motor protein, Kif5B and Usp9X. We observed a dramatic reduction in Kif5B protein levels in polarized Usp9X-null neurons, but not stage 2, suggesting it may play a role in Usp9X’s regulation of axon growth. We are investigating the nature of the Usp9X–Kif5B interaction in axons. As Usp9X has been implicated in neurocognitive and neurodegenerative diseases, such as XLID and Parkinson’s disease, the identification of Usp9X substrates in neurons will be critical to our understanding of the regulation of neuronal function by the ubiquitin pathway. http://dx.doi.org/10.1016/j.ijdevneu.2015.04.066 ISDN2014 0081 Different regulation of AQP4 by fluoxetine in normal and CMS model mice
Reference
Baoman Li 1,2
Stegeman, S., et al., 2013. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFbeta-mediated axonogenesis. PLOS ONE 8 (7), pe68287.
1
http://dx.doi.org/10.1016/j.ijdevneu.2015.04.065
21
Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, University of Rochester Medical Center, United States 2 Department of Clinical Pharmacology, China Medical University, China Fluoxetine, antidepressant drug, is a serotonin-specific reuptake inhibitor (SSRI), the important effects of SSRI for therapy of major depression are the chronic effects, since clinical treatment works slowly, which indicates there should be other drug targets of fluoxetine in the brain. 5-HT2B receptors are necessary for long-term behavioral effects of fluoxetine, which are abolished in 5-HT2B −/− mice or after pharmacologic inactivation of 5-HT2B receptors. We have reported fluoxetine could increase the expres-
increased in the group injected with inhibitor of ERK1/2 , Akt or EGF receptor. In conclusion, Fn has deteriorating effect on the recovery of SCI, which was not only dependent on itself activity, but also attributed to increase the expression of P2Y1 receptor via which ATP caused more astrocytic proliferation and inflammatory response. http://dx.doi.org/10.1016/j.ijdevneu.2015.04.064 ISDN2014 0079 Deubiquitylating enzyme Usp9x regulates neural progenitor fate in vivo Susitha Premarathne ∗ , Mariyam Murtaza, Stephen Wood Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia Neural progenitors (NP) give rise to all the cell types in the brain. During development NPs must balance self-renewal, and hence maintain a stem cell pool, with differentiation into neuron and glia. Regulation of NP fate occurs at multiple levels including post-translational protein modifications by ubiquitylation. Despite the fact that an ubiquitin pathway regulates most aspects of cell function, very little is known of its role in NPs. This is especially true for deubiquitylating enzymes, which remove ubiquitin from proteins and protect them from degradation. Usp9x is a deubiquitylating enzyme and a putative stemness gene highly expressed in NPs. Ectopic expression of Usp9x in embryonic stem cell derived NPs, promotes their self-renewal and polarity. Conversely, conditional in-vivo deletion of Usp9x from mouse NPs resulted in perinatal lethality and disorganisation of the neocortex and allocortex (Stegeman, 2013). Histological and immunoblot analyses of Usp9x-null NPs revealed decreased expression of several definitive adhesion and polarity marker proteins at E12.5. However, most of these markers recovered to normal levels by E14.5. In contrast, the adherens junctional and Wnt signalling molecule, -catenin, showed an increased expression level at both stages. Transcriptomic analysis revealed that the Wnt signalling pathway was significantly increased in the Usp9x-null telencephalon at E14.5. Interestingly, increased -catenin levels were detected in knockout NPs with both decreased (E12.5) and normal (E14.5) levels of N-cadherin suggesting this is an adhesion independent regulatory mechanism. Examination of NP fate at E12.5 revealed a premature differentiation of apical progenitors into abventricularly proliferating Tbr2 positive intermediate NPs. This study for the first time identifies deubiquitylating enzyme mediated post-translational modifications as a novel mechanism of NP fate determination in-vivo.
ISDN2014 0080 Usp9x regulates axon specification and growth M. Tan 1,∗ , L.A. Jolly 2 , M. Murtaza 1 , J. Camonis 3 , S.A. Wood 1 1 Eskitis Institute, Griffith University, Brisbane, Australia 2 SA Pathology, Women’s and Children’s Hospital, North Adelaide, Australia 3 Institute Curie, Paris, France E-mail address: men.tan@griffithuni.edu.au (M. Tan).
Establishing axon-dendrite polarity is essential for unidirectional signal transmission and the formation of complex neural circuits. Defective axon formation can lead to neurodegenerative and neurocognitive diseases. Conditional deletion of the deubiquitylating enzyme, Usp9X results in shorter axons in hippocampal neurons at 3, 5 and 7 days in culture. To study the role of Usp9X in axonal specification we compared wild type and Usp9X-null hippocampal neurons in the first 48 h of culture and categorised them into three stages. Stage 1 neurons extend a prominent lamella around the cell body. Stage 2 neurons extend short symmetrical neurites. During the transition from stages 2 to 3, neurite symmetry is broken and one neurite is specified to become an axon. From 12 h onward Usp9X-null neurons significantly (p = 0.002) lagged behind wild-type in progression from stages 1 to 2. However despite a lag of about 10 h the overall kinetics for stages 1 to 2 progression was similar. A more dramatic effect of Usp9X loss was during stages 2 to 3 progression where Usp9X-null neurons required over 36 h longer to specify an axon (p = 0.001). After this lag Usp9X-null neurons entered stage 3 at a similar rate to wild-type. Therefore Usp9X is required for axon specification. Using yeast-2-hybrid screening we identified an interaction between the kinesin1 motor protein, Kif5B and Usp9X. We observed a dramatic reduction in Kif5B protein levels in polarized Usp9X-null neurons, but not stage 2, suggesting it may play a role in Usp9X’s regulation of axon growth. We are investigating the nature of the Usp9X–Kif5B interaction in axons. As Usp9X has been implicated in neurocognitive and neurodegenerative diseases, such as XLID and Parkinson’s disease, the identification of Usp9X substrates in neurons will be critical to our understanding of the regulation of neuronal function by the ubiquitin pathway. http://dx.doi.org/10.1016/j.ijdevneu.2015.04.066 ISDN2014 0081 Different regulation of AQP4 by fluoxetine in normal and CMS model mice
Reference
Baoman Li 1,2
Stegeman, S., et al., 2013. Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFbeta-mediated axonogenesis. PLOS ONE 8 (7), pe68287.
1
http://dx.doi.org/10.1016/j.ijdevneu.2015.04.065
21
Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, University of Rochester Medical Center, United States 2 Department of Clinical Pharmacology, China Medical University, China Fluoxetine, antidepressant drug, is a serotonin-specific reuptake inhibitor (SSRI), the important effects of SSRI for therapy of major depression are the chronic effects, since clinical treatment works slowly, which indicates there should be other drug targets of fluoxetine in the brain. 5-HT2B receptors are necessary for long-term behavioral effects of fluoxetine, which are abolished in 5-HT2B −/− mice or after pharmacologic inactivation of 5-HT2B receptors. We have reported fluoxetine could increase the expres-