Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold

Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen-glucose deprivation as an in vitro model for ischemia, we show that BDNF-tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation

Co-amplification of the HER2 gene and chromosome 17 centromere: a potential diagnostic pitfall in HER2 testing in breast cancer

Co-amplification of the centromere on chromosome 17 (CEP17) and HER2 can occur in breast cancer. Such aberrant patterns (clusters) on CEP17 can be misleading to calculate the HER2/CEP17 ratio, and thus underreporting of HER2 amplification. We identified 14 breast cancers retrospectively with HER2/CEP17 co-amplification and performed FISH (fluorescence in situ hybridization) with additional chromosome 17 probes (17p11.1-q11.1, 17p11.2-p12, TP53 on 17p13.1, RARA on 17q21.1-3 and TOP2 on 17q21.3-22) to characterize the spanning of the amplicon in these cases. Furthermore, the HER2 status was analyzed by means of HER2 silver in situ hybridization (SISH) and immunohistochemistry (IHC). The co-amplification of HER2/CEP17 was compared between the three institutions. TP53 was eusomic in all cases, 17p11.2-p12 in 79% (11/14), whereas 17p11.1-q11.1 showed chromosomal gain in all cases. RARA was amplified in 10/14 cases (71%) and TOP2 in 3/14 cases (21%). HER2 was amplified with FISH/SISH in all 14 cases. 9/14 tumors were 3+ IHC positive (64%) and 3 cases were 2+ IHC positive. In our cohort the CEP17 amplicon almost always involves the HER2 but not the TOP2 locus. Overall agreement on HER2/CEP17 ratio (when applying ASCO/CAP guidelines) was only 64% (9/14 cases) between the institutions. Discrepant ratios varied from 1.1 to 14.3. The HER2/CEP17 co-amplification is not defined in the ASCO/CAP guidelines, and may result in inaccurate HER2-FISH/SISH status, particularly if only the calculated HER2/CEP17 ratio is reported. It is recommended to report separate CEP17 and HER2 signals in complex HER2/CEP17 pattern

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

The catalytic function of the gephyrin-binding protein IQSEC3 regulates neurotransmitter-specific matching of pre- and post-synaptic structures in primary hippocampal cultures

In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post-synaptic proteins of GABAergic synapses are in part apposed to glutamatergic pre-synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre- and post-synaptic elements. We report here that the IQ motif and Sec7 domain-containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post-synaptic density (PSD) proteins. Over-expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co-expressing eGFP-gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP-gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP-gephyrin, endogenous GABA receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over-expression. Conversely, GFP-PSD-95 clusters were unaffected by over-expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant-negative mutant over-expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca concentrations. Taken together, our findings identify a post-synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre-synaptic terminals

Differential impact of GABAA Receptors on Inhibitory connectivity for Superficial Pyramidal Neuron Responsiveness In Vivo

A diverse set of GABAA receptors (GABAARs) enable synaptic plasticity adaptations at inhibitory postsynaptic sites in collaboration with the scaffolding protein gephyrin. Early studies helped to identify distinctions between GABAAR subsets allocated within specific functional circuits, but their contribution to the changing dynamics of a microcircuit remains unclear. Here, using the whisker-barrel system we assessed the contribution of specific GABAAR subtypes to sensory processing in vivo. We monitored spontaneous and evoked Ca2+ transients in layer 2/3 pyramidal cells with the genetically encoded Ca2+ sensor RCaMP1.07. Using Gabra1 or Gabra2 global and conditional knockout mice, we uncovered that α1- and α2-GABAARs determine the sparseness of L2/3 pyramidal neuron encoding. In a cell-type dependent manner, α1-GABAARs affected neuronal excitability while α2-GABAARs influenced the reliability of neuronal responses after whisker stimulation. We also discerned that gephyrin and its diverse post-translational modifications (PTMs) facilitate microcircuit homeostasis. Our results underscore the relevance of the diversity of GABAARs within a cortical microcircuit

RhoGEF9 splice isoforms influence neuronal maturation and synapse formation downstream of α2 GABAA receptors

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism

RhoGEF9 splice isoforms influence neuronal maturation and synapse formation downstream of alpha 2 GABA(A) receptors

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin ( CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABA(A)Rs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of alpha 2-containing GABA(A)Rs during neuron maturation in a Cdc42 dependent mechanism.Peer reviewe

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation

Abstract Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation

eGFP-CB1_SH3- can rescue gephyrin scaffolding in <i>Gabra2</i> KO neurons.

<p><b>(A)</b> Morphology of retrovirus eGFP with proximal and distal dendritic sites. Right panels; WT and <i>Gabra2</i> KO neuron expressing eGFP with gephyrin and α1 GABA_AR staining. Arrows show reduced gephyrin clustering and increased α1 GABA_AR clusters in <i>Gabra2</i> KO neurons. <b>(B-B’)</b> α1 GABA_AR cluster density in proximal dendritic segments show significant increase in <i>Gabra2</i> KO cells at 14 dpi, 28 dpi, but not 42 dpi. Similarly, α1 GABA_AR cluster density in distal dendritic segments are increased <i>Gabra2</i> KO cells at 28 dpi, but not 14 dpi and 42 dpi. <b>(C-C’)</b> Gephyrin cluster density in proximal and distal dendritic segments show significant reduction in <i>Gabra2</i> KO cells at 14 dpi, 28 dpi and 42 dpi. <b>(D-D’)</b> Gephyrin clustering at both proximal and distal dendritic segments is restored in <i>Gabra2</i> KO neurons upon eGFP-CB1_SH3- overexpression. Rescue of gephyrin clustering does not influence α1 GABA_AR cluster density in <i>Gabra2</i> KO neurons.</p

Protein half-life of V5-CB1 and V5-CB2 splice isoforms differ.

<p><b>(A)</b> Protein stability of V5-CB1_SH3+ was determined by collecting the protein samples at 0, 1, 2, 4 and 8 hours in the presence of cyclohexamide (CHX) (0.1mM) and half-life relative to actin was plotted. The t_1/2 and R² values were determined (inset). <b>(B)</b> Protein half-life of CB2_SH3+ isoform. <b>(C)</b> Protein half-life of CB1_SH3- in HEK-293T cells. <b>(D)</b> Protein half-life of CB2_SH3- isoform in HEK-293T cells. CB1_SH3- shows the shortest half-life (1.8 hr), while CB2_SH3- has the longest half-life (7.294 hr). <b>(E)</b> Protein half-life of mCherry-CB1_SH3- in primary hippocampal neuron is similar to HEK-293T cells. <b>(F)</b> CB1 isoform C-terminal amino acid sequence with specific Ub lysine residues marked in red. <b>(G-J)</b> The predicted K491R/K492R ubiquitin residues in CB1 were mutated Lys/Arg and the protein stability of the mutants was determined. The ubiquitin site mutants have a longer half-life compared to the WT counterparts. <b>(I-J)</b> CBΔC_SH3+ and CBΔC_SH3- C-terminus deletion mutations show similar protein half-life.</p