Distinct functional modes of SUMOylation for retinoid X receptor alpha.

The present study investigated human retinoid X receptor alpha (hRXRα) as a substrate for modification with small ubiquitin like modifier (SUMO) and how members of the protein inhibitor of activated STAT (PIAS) family may impact upon this process. In agreement with a previous study, we validate Ubc9 to facilitate SUMOylation of hRXRα at lysine 108 but note this modification to occur for all isoforms rather than specifically with SUMO1 and to preferentially occur with the unliganded form of hRXRα. SUMOylation of hRXRα is significantly enhanced through PIAS4-mediated activity with lysine 245 identified as a specific SUMO2 acceptor site modified in a PIAS4-dependent fashion. While individual mutations at lysine 108 or 245 modestly increase receptor activity, the combined loss of SUMOylation at both sites significantly potentiates the transcriptional responsiveness of hRXRα suggesting both sites may cooperate in a DNA element-dependent context. Our findings highlight combinatorial effects of SUMOylation may regulate RXRα-directed signalling in a gene-specific fashion.

The neural and peptidergic control of gut contraction in Locusta migratoria: the effect of an FGLa/AST.

The regulation of insect gut physiology is complex and involves the interactions of a number of mechanisms, including the neural regulation of gut contraction by altering neural input and the modulation of gut contractions by neuropeptides directly affecting the muscle. The FGLa-type allatostatins (FGLa/ASTs) are known brain/gut peptides with numerous physiological roles, including modulation of gut contraction and neural input. To further investigate the pleiotropic roles of FGLa/AST peptides in Locusta migratoria, we have examined the role of a locust FGLa/AST (Scg-AST-6) in the gut. Proctolin and Scg-AST-6 have opposing effects on gut contraction, where proctolin dose-dependently increases gut muscle tension, while Scg-AST-6 inhibits both muscle tension and spontaneous and neurogenic contractions in a dose-dependent manner. Results from neurophysiological recordings indicate that there may be a central pattern generator (CPG) within the ventricular ganglia regulated by descending inhibition, and the addition of Scg-AST-6 dose-dependently modulates this ventricular ganglion CPG. This work provides a comprehensive picture of how FGLa/ASTs may modulate and coordinate each region of the locust gut, and shows that FGLa/ASTs have both central effects, on the ventricular ganglion CPG, and peripheral effects on the gut muscle. Overall, this study shows how FGLa/ASTs contribute to the complex regulation and fine tuning of gut contraction.

Structural determinants of transmission at large hippocampal mossy fiber synapses.

Synapses are the key elements for signal processing and plasticity in the brain. To determine the structural factors underlying the unique functional properties of the hippocampal mossy fiber synapse, the complete quantitative geometry was investigated, using electron microscopy of serial ultrathin sections followed by computer-assisted three-dimensional reconstruction. In particular, parameters relevant for transmitter release and synaptic plasticity were examined. Two membrane specializations were found: active zones (AZs), transmitter release sites, and puncta adherentia, putative adhesion complexes. Individual boutons had, on average, 25 AZs (range, 7-45) that varied in shape and size (mean, 0.1 microm2; range, 0.07-0.17 microm2). The mean distance between individual AZs was 0.45 microm. Mossy fiber boutons and their target structures were mostly ensheathed by astrocytes, but fine glial processes never reached the active zones. Two structural factors are likely to promote synaptic cross talk: the short distance between AZs and the absence of fine glial processes at AZs. Thus, synaptic cross talk may contribute to the efficacy of hippocampal mossy fiber synapses. On average, a bouton contained 20,400 synaptic vesicles; approximately 900 vesicles were located within 60 nm from the active zone, approximately 4400 between 60 and 200 nm, and the remaining beyond 200 nm, suggesting large readily releasable, recycling, and reserve pools. The organization of the different pools may be a key structural correlate of presynaptic plasticity at this synapse. Thus, the mossy fiber bouton differs fundamentally in structure and function from the calyx of Held and other central synapses.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing.

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

Structural plasticity of spines at giant mossy fiber synapses.

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.