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RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein ( Arc ) and inositol 1,4,5-trisphosphate receptor type 1 ( Ip3r1 ) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Fujita et al.)

We examined the transcriptional regulation of the activity-regulated cytoskeleton-associated protein gene (Arc), focusing on BDNF-induced Arc expression in cultured rat cortical cells. Although the synaptic activity-responsive element (SARE), located -7 kbp upstream of the Arc transcription start site, responded to NMDA, BDNF, or FGF2, the proximal region of the promoter (Arc/-1679) was activated by BDNF or FGF2, but not by NMDA, suggesting the presence of at least two distinct Arc promoter regions, distal and proximal, that respond to extracellular stimuli. Specificity protein 4 (SP4) and early growth response 1 (EGR1) controlled Arc/-1679 transcriptional activity via the region encompassing -169 to -37 of the Arc promoter. We found that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, significantly enhanced the inductive effects of BDNF or FGF2, but not those of NMDA on Arc expression. Inhibitors of class I/IIb HDACs, SAHA, and class I HDACs, MS-275, but not of class II HDACs, MC1568, enhanced BDNF-induced Arc expression. The enhancing effect of TSA was mediated by the region from -1027 to -1000 bp, to which serum response factor (SRF) and HDAC1 bound. The binding of HDAC1 to this region was reduced by TSA. Thus, Arc expression was suppressed by class I HDAC-mediated mechanisms via chromatin modification of the proximal promoter whereas the inhibition of HDAC allowed Arc expression to be markedly enhanced in response to BDNF or FGF2. These results contribute to our understanding of the physiological role of Arc expression in neuronal functions such as memory consolidation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Synaptic and extrasynaptic NMDA receptors (NMDARs) appear to play opposite roles in neuronal survival and death. Here we report the new findings on the dysregulation of survival factor, myocyte enhancer factor 2D (MEF2D), by extrasynaptic NMDARs. Excitotoxicity led to the NMDAR-dependent degradation of MEF2D protein and inhibition of its transactivation activity in mature cortical neurons. The activation of extrasynaptic NMDARs alone was sufficient for degradation of MEF2D. Calpain directly cleaved MEF2D in vitro and blocking this protease activity greatly attenuated NMDAR signaled degradation of MEF2D in neurons. Consistently, inhibition of calpain protected cortical neurons from NMDA-induced excitotoxicity. Furthermore, knockdown of MEF2D sensitized neurons to NMDA-induced excitotoxicity, which was not protected by calpain inhibition. Collectively, these findings suggest that dysregulation of MEF2D by calpain may mediate excitotoxicity via an extrasynaptic NMDAR-dependent manner.

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

During a screen for novel putative Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like CREB kinases (CLICKs), we have cloned a full-length cDNA for CLICK-III/CaMKIgamma, an isoform of the CaMKI family with an extended C-terminal domain ending with CAAX motif (where AA is aliphatic acid). As expected from the similarity of its kinase domain with the other CaMKI isoforms, full activation of CLICK-III/CaMKIgamma required both Ca(2+)/CaM and phosphorylation by CaMKK. We also found that Ca(2+)/cAMP-response element-binding protein (CREB) was a good substrate for CLICK-III/CaMKIgamma, at least in vitro. Interestingly enough, CLICK-III/CaMKIgamma transcripts were most abundant in neurons, with the highest levels in limited nuclei such as the central nucleus of the amygdala (CeA) and the ventromedial hypothalamus. Consistent with the presence of the CAAX motif, CLICK-III/CaMKIgamma was found to be anchored to various membrane compartments, especially to Golgi and plasma membranes. Both point mutation in the CAAX motif and treatment with compactin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted such membrane localization, suggesting that membrane localization of CLICK-III/CaMKIgamma occurred in a prenylation-dependent way. These findings provide a novel mechanism by which neuronal CaMK activity could be targeted to specific membrane compartments.