The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.

BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

Quantitative Ubiquitylome Analysis Reveals the Specificity of RNF111/Arkadia E3 Ubiquitin Ligase for its Degradative Substrates SKI and SKIL/SnoN in TGF-ß Signaling Pathway.

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-ß (TGF-ß) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-ß signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-ß signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-ß. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-ß pathway activation.

Trans-Synaptic Signaling through the Glutamate Receptor Delta-1 Mediates Inhibitory Synapse Formation in Cortical Pyramidal Neurons.

Fine orchestration of excitatory and inhibitory synaptic development is required for normal brain function, and alterations may cause neurodevelopmental disorders. Using sparse molecular manipulations in intact brain circuits, we show that the glutamate receptor delta-1 (GluD1), a member of ionotropic glutamate receptors (iGluRs), is a postsynaptic organizer of inhibitory synapses in cortical pyramidal neurons. GluD1 is selectively required for the formation of inhibitory synapses and regulates GABAergic synaptic transmission accordingly. At inhibitory synapses, GluD1 interacts with cerebellin-4, an extracellular scaffolding protein secreted by somatostatin-expressing interneurons, which bridges postsynaptic GluD1 and presynaptic neurexins. When binding to its agonist glycine or D-serine, GluD1 elicits non-ionotropic postsynaptic signaling involving the guanine nucleotide exchange factor ARHGEF12 and the regulatory subunit of protein phosphatase 1 PPP1R12A. Thus, GluD1 defines a trans-synaptic interaction regulating postsynaptic signaling pathways for the proper establishment of cortical inhibitory connectivity and challenges the dichotomy between iGluRs and inhibitory synaptic molecules.

Microglia control the glycinergic but not the GABAergic synapses via prostaglandin E2 in the spinal cord.

Microglia control excitatory synapses, but their role in inhibitory neurotransmission has been less well characterized. Herein, we show that microglia control the strength of glycinergic but not GABAergic synapses via modulation of the diffusion dynamics and synaptic trapping of glycine (GlyR) but not GABAA receptors. We further demonstrate that microglia regulate the activity-dependent plasticity of glycinergic synapses by tuning the GlyR diffusion trap. This microglia-synapse cross talk requires production of prostaglandin E2 by microglia, leading to the activation of neuronal EP2 receptors and cyclic adenosine monophosphate-dependent protein kinase. Thus, we now provide a link between microglial activation and synaptic dysfunctions, which are common early features of many brain diseases.

NOS2 expression is restricted to neurons in the healthy brain but is triggered in microglia upon inflammation.

Nitric oxide (NO) is a diffusible second messenger with a great variety of functions in the brain. NO is produced by three isoforms of NO synthase (NOS), NOS1, NOS2, and NOS3. Although broad agreement exists regarding the expression of NOS1 and NOS3 in neurons and endothelial cells, the pattern of NOS2 expression is still controversial and remains elusive. We have now generated a novel transgenic mouse that expresses the fluorescent reporter tdTomato and the CRE recombinase under the control of the Nos2 gene regulatory regions. Such tool allows the reliable tracking of NOS2 expression in tissue and further unravels episodes of transient NOS2 expression. Using this transgenic mouse, we show that in the healthy brain, NOS2 is only transiently expressed in neurons scattered in the piriform and entorhinal cortex, the amygdaloid nuclei, the medial part of the thalamus, the hypothalamus, the dentate gyrus, and the cerebellum. NOS2 expression was rarely detected in microglia. We further show that inflammation, induced by intracerebral injection of LPS and IFNγ, triggers transient expression of NOS2 in microglia but not in neurons. This novel transgenic tool has thus allowed us to clarify the NOS2 expression pattern and its differential profile in neurons and microglia in healthy and inflammatory conditions.

Microglia shape corpus callosum axon tract fasciculation: functional impact of prenatal inflammation.

Microglia colonise the brain parenchyma at early stages of development and accumulate in specific regions where they participate in cell death, angiogenesis, neurogenesis and synapse elimination. A recurring feature of embryonic microglial is their association with developing axon tracts, which, together with in vitro data, supports the idea of a physiological role for microglia in neurite development. Yet the demonstration of this role of microglia is lacking. Here, we have studied the consequences of microglial dysfunction on the formation of the corpus callosum, the largest commissure of the mammalian brain, which shows consistent microglial accumulation during development. We studied two models of microglial dysfunction: the loss-of-function of DAP12, a key microglial-specific signalling molecule, and a model of maternal inflammation by peritoneal injection of lipopolysaccharide at embryonic day (E)15.5. We also took advantage of the Pu.1(-/-) mouse line, which is devoid of microglia. We performed transcriptional profiling of maternally inflamed and Dap12-mutant microglia at E17.5. The two treatments principally down-regulated genes involved in nervous system development and function, particularly in neurite formation. We then analysed the developmental consequences of these microglial dysfunctions on the formation of the corpus callosum. We show that all three models of altered microglial activity resulted in the defasciculation of dorsal callosal axons. Our study demonstrates that microglia display a neurite-development-promoting function and are genuine actors of corpus callosum development. It further shows that microglial activation impinges on this function, thereby revealing that prenatal inflammation impairs neuronal development through a loss of trophic support.

Depletion of microglia from primary cellular cultures.

Primary cultures are an important in vitro tool to study cellular processes and interactions. These cultures are complex systems, composed of many cell types, including neurons, astrocytes, oligodendrocytes, microglia, NG2 cells, and endothelial cells. For some studies it is necessary to be able to study a pure culture of one cell type, or eliminate a particular cell type, to better understand its function. There exist cell culture protocols for making pure astrocyte or microglia cultures. Here we present two protocols to produce cultures depleted for microglia: in the first case, from a mixed astrocyte-microglia culture and, in the second, for eliminating microglia from neuronal cultures.

Differential control of thrombospondin over synaptic glycine and AMPA receptors in spinal cord neurons.

Thrombospondin-1 (TSP-1) is a large extracellular matrix protein secreted by astrocytes during development and inflammation. In the developing CNS, TSP-1 is involved in neuronal migration and adhesion, neurite outgrowth, and synaptogenesis. We investigated the effects of TSP-1 on neurons with mature synapses using immunocytochemistry, single-particle tracking, surface biotinylation, and calcium imaging. We show that in cultured rat spinal cord neurons TSP-1 decreased neuronal excitability by reducing the accumulation of excitatory AMPA receptors (AMPARs) and increasing that of inhibitory glycine receptors (GlyRs) in synapses. The effects of TSP-1 on GlyRs were dependent on the activation of excitatory receptors. These changes were abolished by blocking ß1-integrins and mimicked by blocking ß3-integrins. In the presence of TSP-1, AMPARs were less stabilized at synapses, increasing their lateral diffusion and endocytosis. Interestingly, TSP-1 counteracted the increased neuronal excitability and neuronal death induced by TNFα. These results suggest a role of TSP-1 in controlling the balance between excitation and inhibition which could help the recovery of normal synaptic activity after injury responses.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression.

A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

The loss of the snoRNP chaperone Nopp140 from Cajal bodies of patient fibroblasts correlates with the severity of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease caused by reduced survival motor neuron (SMN) levels. The assembly machinery containing SMN is implicated in the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN is present in both the cytoplasm and nucleus, where it transiently accumulates in subnuclear domains named Cajal bodies (CBs) and functions in the maturation of snRNPs and small nucleolar (sno)RNPs. The impact of lowering SMN levels on the composition of CBs in SMA cells is still not completely understood. Here, we analyse the CB composition in immortalized and primary fibroblasts from SMA patients. We show that the U snRNA export factors PHAX and chromosome region maintenance 1 and the box C/D snoRNP core protein fibrillarin concentrate in CBs from SMA cells, whereas the box H/ACA core proteins GAR1 and NAP57/dyskerin show reduced CB localization. Remarkably, the functional deficiency in SMA cells is associated with decreased localization of the snoRNP chaperone Nopp140 in CBs that correlates with disease severity. Indeed, RNA interference knockdown experiments in control fibroblasts demonstrate that SMN is required for accumulation of Nopp140 in CBs. Conversely, overexpression of SMN in SMA cells restores the CB localization of Nopp140, whereas SMN mutants found in SMA patients are defective in promoting the association of Nopp140 with CBs. Taken together, we demonstrate that only a subset of CB functions (as indicated by the association of representative factors) are impaired in SMA cells and, importantly, we identify the decrease of Nopp140 localization in CBs as a phenotypic marker for SMA.