Disrupted in schizophrenia 1 (DISC1) L100P mutants have impaired activity-dependent plasticity in vivo and in vitro

Major neuropsychiatric disorders are genetically complex but share overlapping etiology. Mice mutant for rare, highly penetrant risk variants can be useful in dissecting the molecular mechanisms involved. The gene disrupted in schizophrenia 1 (DISC1) has been associated with increased risk for neuropsychiatric conditions. Mice mutant for Disc1 display morphological, functional and behavioral deficits that are consistent with impairments observed across these disorders. Here we report that Disc1 L100P mutants are less able to reorganize cortical circuitry in response to stimulation in vivo. Molecular analysis reveals that the mutants have a reduced expression of PSD95 and pCREB in visual cortex and fail to adjust expression of such markers in response to altered stimulation. In vitro analysis shows that mutants have impaired functional reorganization of cortical neurons in response to selected forms of neuronal stimulation, but there is no altered basal expression of synaptic markers. These findings suggest that DISC1 has a critical role in the reorganization of cortical plasticity and that this phenotype becomes evident only under challenge, even at early postnatal stages. This result may represent an important etiological mechanism in the emergence of neuropsychiatric disorders

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain

SUMOylation and calcium signalling: potential roles in the brain and beyond

Small ubiquitin-like modi er (SUMO) conjugation (or SUMOylation) is a post-translational protein modi cation implicated in alterations to protein expression, localization and func- tion. Despite a number of nuclear roles for SUMO being well characterized, this process has only started to be explored in relation to membrane proteins, such as ion channels. Cal- cium ion (Ca2+) signalling is crucial for the normal functioning of cells and is also involved in the pathophysiological mechanisms underlying relevant neurological and cardiovascu- lar diseases. Intracellular Ca2+ levels are tightly regulated; at rest, most Ca2+ is retained in organelles, such as the sarcoplasmic reticulum, or in the extracellular space, whereas depolarization triggers a series of events leading to Ca2+ entry, followed by extrusion and reuptake. The mechanisms that maintain Ca2+ homoeostasis are candidates for modulation at the post-translational level. Here, we review the effects of protein SUMOylation, including Ca2+ channels, their proteome and other proteins associated with Ca2+ signalling, on vital cellular functions, such as neurotransmission within the central nervous system (CNS) and in additional systems, most prominently here, in the cardiac system

Investigating the molecular pathways driving the sumoylation/desumoylation balance in rat hippocampal synapses

La SUMOylation est une modification post-traductionnelle essentielle pour toutes les cellules eucaryotes. C’est un processus enzymatique qui permet la liaison covalente du polypeptide SUMO sur des résidus lysine de protéines cibles. La SUMOylation est un processus réversible sous l’action de désumoylases appelées SENP. Il est critique de maintenir un équilibre entre forme modifiée et non modifiée d’un substrat donné. En effet, la dérégulation de la balance SUMOylation/déSUMOylation a été mise en évidence dans plusieurs pathologies cérébrales. La synapse est le point de contact entre les neurones où s’effectue la communication synaptique. Ce sont des structures très denses où le processus de SUMOylation régule l’interaction et la fonction de multiples protéines. Durant ma thèse, j'ai combiné l’utilisation de l'imagerie en temps réel sur cellules vivantes avec des approches biochimiques et pharmacologiques pour identifier les mécanismes de régulation du transport de SENP1. J'ai ainsi démontré que l'activation neuronale augmente les niveaux synaptiques de SENP1. Cette augmentation synaptique résulte de la modification de la vitesse de diffusion de l’enzyme SENP1 entre les dendrites et les synapses d’une part, et d’autre part, de l’augmentation importante du temps de rétention synaptique de l’enzyme. Je rapporte également que ce mécanisme de régulation dynamique de SENP1 implique l'activation des récepteurs métabotropiques du glutamate. De plus, je suggère la participation du processus de phosphorylation dans cette régulation synapto-dendritique de SENP1 mettant ainsi en lumière un nouveau mécanisme de régulation de la balance neuronale entre SUMOylation et déSUMOylation.Sumoylation is a vital eukaryotic posttranslational modification. Sumoylation occurs as an enzymatic cycle that conjugates SUMO proteins to target proteins. SUMO proteases (SENP) deconjugate SUMO from modified proteins and thus maintain balanced levels of SUMOylated and un-SUMOylated proteins required for physiological homeostasis. Neuronal synapses are protein-rich structures that underlie synaptic transmission and plasticity. Strong evidence exists that sumoylation occurs in synapses and regulates the function of synaptic proteins. Indeed, distortion of the SUMO balance has been linked to several pathologies of the synapse. Gaining a deeper understanding into the molecular mechanisms regulating the SUMO balance is a prerequisite to envisaging the development of novel therapies. In my PhD work, I used a combination of live-cell confocal imaging, protein biochemistry and pharmacological approaches to identify SENP1 regulatory mechanisms at synapses. I provided evidence that synaptic activation increases SENP1 protein levels at synapses. I showed that the increase in synaptic SENP1 upon synaptic activation is a result of two processes: Although (a) fewer SENP1 proteins enter into spines at low diffusion speed (b) a significant proportion of SENP1 becomes immobile and is retained in spines. I demonstrate that the regulatory mechanisms of SENP1 dynamics involve a direct activation of mGlu1/5 receptors. Moreover, I suggest that phosphorylation may play an important regulatory role in SENP1 synapto-dendritic diffusion. Altogether, I propose a novel mechanism driving for the SUMO balance at synapses

Étude des mécanismes de régulation synaptique de la balance sumoylation/désumoylation

Sumoylation is a vital eukaryotic posttranslational modification. Sumoylation occurs as an enzymatic cycle that conjugates SUMO proteins to target proteins. SUMO proteases (SENP) deconjugate SUMO from modified proteins and thus maintain balanced levels of SUMOylated and un-SUMOylated proteins required for physiological homeostasis. Neuronal synapses are protein-rich structures that underlie synaptic transmission and plasticity. Strong evidence exists that sumoylation occurs in synapses and regulates the function of synaptic proteins. Indeed, distortion of the SUMO balance has been linked to several pathologies of the synapse. Gaining a deeper understanding into the molecular mechanisms regulating the SUMO balance is a prerequisite to envisaging the development of novel therapies. In my PhD work, I used a combination of live-cell confocal imaging, protein biochemistry and pharmacological approaches to identify SENP1 regulatory mechanisms at synapses. I provided evidence that synaptic activation increases SENP1 protein levels at synapses. I showed that the increase in synaptic SENP1 upon synaptic activation is a result of two processes: Although (a) fewer SENP1 proteins enter into spines at low diffusion speed (b) a significant proportion of SENP1 becomes immobile and is retained in spines. I demonstrate that the regulatory mechanisms of SENP1 dynamics involve a direct activation of mGlu1/5 receptors. Moreover, I suggest that phosphorylation may play an important regulatory role in SENP1 synapto-dendritic diffusion. Altogether, I propose a novel mechanism driving for the SUMO balance at synapses.La SUMOylation est une modification post-traductionnelle essentielle pour toutes les cellules eucaryotes. C’est un processus enzymatique qui permet la liaison covalente du polypeptide SUMO sur des résidus lysine de protéines cibles. La SUMOylation est un processus réversible sous l’action de désumoylases appelées SENP. Il est critique de maintenir un équilibre entre forme modifiée et non modifiée d’un substrat donné. En effet, la dérégulation de la balance SUMOylation/déSUMOylation a été mise en évidence dans plusieurs pathologies cérébrales. La synapse est le point de contact entre les neurones où s’effectue la communication synaptique. Ce sont des structures très denses où le processus de SUMOylation régule l’interaction et la fonction de multiples protéines. Durant ma thèse, j'ai combiné l’utilisation de l'imagerie en temps réel sur cellules vivantes avec des approches biochimiques et pharmacologiques pour identifier les mécanismes de régulation du transport de SENP1. J'ai ainsi démontré que l'activation neuronale augmente les niveaux synaptiques de SENP1. Cette augmentation synaptique résulte de la modification de la vitesse de diffusion de l’enzyme SENP1 entre les dendrites et les synapses d’une part, et d’autre part, de l’augmentation importante du temps de rétention synaptique de l’enzyme. Je rapporte également que ce mécanisme de régulation dynamique de SENP1 implique l'activation des récepteurs métabotropiques du glutamate. De plus, je suggère la participation du processus de phosphorylation dans cette régulation synapto-dendritique de SENP1 mettant ainsi en lumière un nouveau mécanisme de régulation de la balance neuronale entre SUMOylation et déSUMOylation

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain

The synaptic balance between sumoylation and desumoylation is maintained by the activation of metabotropic mGlu5 receptors

International audienceSumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions

Bidirectional regulation of synaptic SUMOylation by Group 1 metabotropic glutamate receptors

International audienceSUMOylation is a post-translational modification essential to cell homeostasis. A tightly controlled equilibrium between SUMOylation and deSUMOylation processes is also critical to the neuronal function including neurotransmitter release and synaptic transmission and plasticity. Disruption of the SUMOylation homeostasis in neurons is associated with several neurological disorders. The balance between the SUMOylation and deSUMOylation of substrate proteins is maintained by a group of deSUMOylation enzymes called SENPs. We previously showed that the activation of type 5 metabotropic glutamate receptors (mGlu5R) first triggers a rapid increase in synaptic SUMOylation and then upon the sustained activation of these receptors, the deSUMOylase activity of SENP1 allows the increased synaptic SUMOylation to get back to basal levels. Here, we combined the use of pharmacological tools with subcellular fractionation and live-cell imaging of individual hippocampal dendritic spines to demonstrate that the synaptic accumulation of the deSUMOylation enzyme SENP1 is bidirectionally controlled by the activation of type 1 mGlu1 and mGlu5 receptors. Indeed, the pharmacological blockade of mGlu1R activation during type 1 mGluR stimulation leads to a faster and greater accumulation of SENP1 at synapses indicating that mGlu1R acts as a brake to the mGlu5R-dependent deSUMOylation process at the post-synapse. Altogether, our findings reveal that type 1 mGluRs work in opposition to dynamically tune the homeostasis of SUMOylation at the mammalian synapse