Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

BACKGROUND: Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants. RESULTS: In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs. CONCLUSION: A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR

BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for BDNF-live-exon-visualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications

Identifikation eines putativen SUT1 RNA-bindenden Proteins durch die "Yeast Three Hybrid" Methode

Sucrose is the major nutrient product of the photosynthesis reactions; its correct distribution via the specialized cells of the phloem is essential to plant survival. The transport of sucrose within solanaceous plants is mediated in part by sucrose transport proteins (SUTs) localized to the plasma membrane of the phloem sieve elements (SEs). SUT1, which is essential for phloem loading of sucrose, shows the predicted localization to the enucleate SE; its mRNA, initially thought to be found exclusively in the closely associated companion cell (CC), is also found in the enucleate SE. The mechanism of arrival and purpose of this mRNA localization in solanaceous plants has become a matter of dispute. The present work deals with the application of an in vivo method for the discovery of mRNA binding proteins, and the identification of a putative LeSUT1 mRNA binding protein. A high quality cDNA library encoding tomato leaf protein fusions was synthesized and cloned for use in the yeast three-hybrid method (SenGupta et al., 1996). Several RNA hybrid molecules were designed based on the sequence and secondary structure of LeSUT1 mRNA and used to screen the cDNA library for putative mRNA binding proteins. A single isolated cDNA clone was shown to interact with two structurally dissimilar RNA baits. Northern blot and RACE analysis suggest that the isolated cDNA is a fragment of a larger molecule; sequence analysis suggests the presence of a putative RNA binding domain. The isolated cDNA encodes a novel protein molecule that is able to specifically interact with LeSUT1 mRNA in vivo and may be involved in the localization of SUT1 mRNA. While the localization of SUT1 protein was previously established (Kühn et al., 1997), the mechanism of this localization has yet to be identified. In order to examine this process, tobacco sections were exposed to the fungal toxin Brefeldin A (BFA), which inhibits anterograde vesicle transport of proteins. SUT1 protein localization was inhibited by BFA application to fresh sections in preliminary experiments. When fresh tobacco sections were incubated in water, or exposed to no treatment, SUT1 protein was predictably localized to the SE. When fresh sections were exposed to 50 mM BFA for 10 minutes, however, SUT1 protein was sequestered in the CC; was visibly increased upon exposure to 100 mM BFA. These initial results contribute to the formation of an emerging model of SUT1 localization in which protein and mRNA are localized in separate but related events. The highly determined secondary structure of LeSUT1 3'UTR as well as the existence of multiple polyadenylation states also serves to support the concept of a highly regulated complex system resulting in the localization of SUT1 to the plasma membrane of the enucleate SE.Saccharose ist das Hauptprodukt der Photosynthese und die richtige Verteilung von Saccharose durch das Phloem ist lebenswichtig für alle Pflanzen. Der Saccharosetransport in Solanaceen wird zum Teil durch Saccharosetransporter (SUT) vermittelt, die in der Plasmamembran der Siebelemente (SE) lokalisiert sind. SUT1, welches wesentlich zur Phloembeladung mit Saccharose beiträgt, ist wie erwartet in den zellkernlosen Siebelementen lokalisiert. Die SUT1 mRNA, die ursprünglich ausschließlich in den danebenliegenden Geleitzellen (CC) vermutet wurde, wird ebenfalls im zellkernlosen SE gefunden. Die Frage, wie SUT1 mRNA dorthin gelangt, sowie der Zweck dieser Lokalisierung in SE der Solanaceen ist noch ungeklärt. Die vorliegende Arbeit befasst sich mit der Anwendung einer in vivo Methode zur Suche nach mRNA-bindenden Proteinen sowie mit der Identifizierung eines möglichen LeSUT1 mRNA-bindenden Proteins. Eine qualitativ hochwertige cDNA Bibliothek aus Tomatenblättern zur Anwendung in die 'Yeast three-hybrid' Methode wurde hergestellt. Basierend auf der Sequenz und der Sekundärstruktur von LeSUT1 mRNA wurden mehrere RNA Hybridmoleküle entworfen und als 'bait' zum 'screening' der cDNA Bibliothek nach möglichen mRNA-bindenden Proteinen verwendet. Ein einziger cDNA Klon interagierte mit zwei in ihrer Struktur unterschiedlichen RNA Hybridmolekülen. Northern Blot und RACE Experimente lassen vermuten, dass die isolierte cDNA ein Fragment eines größeren Moleküls darstellt. Die Sequenzanalyse dieses Fragments zeigt eine mögliche RNA-bindende Domäne. Die isolierte cDNA codiert für ein unbekanntes Protein, welches eine spezifische Interaktion mit der LeSUT1 mRNA zeigt und möglicherweise an der Lokalisierung der SUT1 mRNA beteiligt ist. Obwohl die Lokalisation von SUT1 Protein bereits gezeigt wurde (Kühn et al. 1997), ist der Lokalisierungsmechanismus noch nicht geklärt. Um diesen Mechanismus zu untersuchen wurden frische Tabakschnitte mit dem Toxin Brefeldin A (BFA) behandelt, welches den Vesikeltransport von Proteinen hemmt. In vorausgehenden Experimenten inhibierte die BFA-Behandlung die Lokalisierung von SUT1 Protein. Die Inkubation von Tabakschnitten in Wasser sowie gar keine Behandlung der Schnitte führte zur Lokalisierung von SUT1 Protein in SE. Im Gegensatz dazu führte die Behandlung der Tabakschnitte mit BFA (50 mM BFA, 10 min) zur Akkumulation von SUT1 Protein in den Geleitzellen. Dieser Effekt wurde deutlich verstärkt durch die Behandlung mit 100 mM BFA. Diese ersten Ergebnisse tragen zur Erstellung eines sich entwickelnden Modells der SUT1 Lokalisierung bei, in welchem Protein und mRNA in getrennten aber in Beziehung zueinander stehenden Ereignissen lokalisiert werden. Sowohl die klar vorausgesagt Sekundärstruktur von LeSUT1 3'UTR als auch das Vorkommen von mehrfachen Polyadenylationstypen unterstützen ebenfalls die Vorstellung eines hochregulierten komplexen Systems, welches in der Lokalisierung von SUT1 an der Plasmamembran des zellkernlosen SE resultiert

The reduced cochlear output and the failure to adapt the central auditory response causes tinnitus in noise exposed rats.

Tinnitus is proposed to be caused by decreased central input from the cochlea, followed by increased spontaneous and evoked subcortical activity that is interpreted as compensation for increased responsiveness of central auditory circuits. We compared equally noise exposed rats separated into groups with and without tinnitus for differences in brain responsiveness relative to the degree of deafferentation in the periphery. We analyzed (1) the number of CtBP2/RIBEYE-positive particles in ribbon synapses of the inner hair cell (IHC) as a measure for deafferentation; (2) the fine structure of the amplitudes of auditory brainstem responses (ABR) reflecting differences in sound responses following decreased auditory nerve activity and (3) the expression of the activity-regulated gene Arc in the auditory cortex (AC) to identify long-lasting central activity following sensory deprivation. Following moderate trauma, 30% of animals exhibited tinnitus, similar to the tinnitus prevalence among hearing impaired humans. Although both tinnitus and no-tinnitus animals exhibited a reduced ABR wave I amplitude (generated by primary auditory nerve fibers), IHCs ribbon loss and high-frequency hearing impairment was more severe in tinnitus animals, associated with significantly reduced amplitudes of the more centrally generated wave IV and V and less intense staining of Arc mRNA and protein in the AC. The observed severe IHCs ribbon loss, the minimal restoration of ABR wave size, and reduced cortical Arc expression suggest that tinnitus is linked to a failure to adapt central circuits to reduced cochlear input

BDNF in Lower Brain Parts Modifies Auditory Fiber Activity to Gain Fidelity but Increases the Risk for Generation of Central Noise After Injury

For all sensory organs, the establishment of spatial and temporal cortical resolution is assumed to be initiated by the first sensory experience and a BDNF-dependent increase in intracortical inhibition. To address the potential of cortical BDNF for sound processing, we used mice with a conditional deletion of BDNF in which Cre expression was under the control of the Pax2 or TrkC promoter. BDNF deletion profiles between these mice differ in the organ of Corti (BDNF $^{Pax2}$ -KO) versus the auditory cortex and hippocampus (BDNF $^{TrkC}$ -KO). We demonstrate that BDNF $^{Pax2}$ -KO but not BDNF $^{TrkC}$ -KO mice exhibit reduced sound-evoked suprathreshold ABR waves at the level of the auditory nerve (wave I) and inferior colliculus (IC) (wave IV), indicating that BDNF in lower brain regions but not in the auditory cortex improves sound sensitivity during hearing onset. Extracellular recording of IC neurons of BDNF $^{Pax2}$ mutant mice revealed that the reduced sensitivity of auditory fibers in these mice went hand in hand with elevated thresholds, reduced dynamic range, prolonged latency, and increased inhibitory strength in IC neurons. Reduced parvalbumin-positive contacts were found in the ascending auditory circuit, including the auditory cortex and hippocampus of BDNF $^{Pax2}$ -KO, but not of BDNF $^{TrkC}$ -KO mice. Also, BDNF $^{Pax2}$ -WT but not BDNF $^{Pax2}$ -KO mice did lose basal inhibitory strength in IC neurons after acoustic trauma. These findings suggest that BDNF in the lower parts of the auditory system drives auditory fidelity along the entire ascending pathway up to the cortex by increasing inhibitory strength in behaviorally relevant frequency regions. Fidelity and inhibitory strength can be lost following auditory nerve injury leading to diminished sensory outcome and increased central noise

Cochlear NMDA Receptors as a Therapeutic Target of Noise-Induced Tinnitus

Background: Accumulating evidence suggests that tinnitus may occur despite normal auditory sensitivity, probably linked to partial degeneration of the cochlear nerve and damage of the inner hair cell (IHC) synapse. Damage to the IHC synapses and deafferentation may occur even after moderate noise exposure. For both salicylate- and noise-induced tinnitus, aberrant N-methyl-d-aspartate (NMDA) receptor activation and related auditory nerve excitation have been suggested as origin of cochlear tinnitus. Accordingly, NMDA receptor inhibition has been proposed as a pharmacologic approach for treatment of synaptopathic tinnitus. Methods: Round-window application of the NMDA receptor antagonist AM-101 (Esketamine hydrochloride gel; Auris Medical AG, Basel, Switzerland) was tested in an animal model of tinnitus induced by acute traumatic noise. The study included the quantification of IHC ribbon synapses as a correlate for deafferentation as well as the measurement of the auditory brainstem response (ABR) to close-threshold sensation level stimuli as an indication of sound-induced auditory nerve activity. Results: We have shown that AM-101 reduced the trauma-induced loss of IHC ribbons and counteracted the decline of ABR wave I amplitude generated in the cochlea/auditory nerve. Conclusion: Local round-window application of AM-101 may be a promising therapeutic intervention for the treatment of synaptopathic tinnitus