Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 ...(PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3β, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain.
BackgroundWhile the introduction of immuno-oncology drugs, especially anti-PD-(L)1s, has revolutionized the strategy for fighting against cancer, there is still a huge demand for the development of ...novel anti-tumor drugs from the majority of patients who exhibit refractory or no response to the current immunotherapy options. Meanwhile, cytokine therapy, such as recombinant IL-2, is largely limited due to its high dose-associated severe adverse effects. To overcome this limitation, a novel therapeutic drug which can activate immune cells with higher specificity and stronger potency should be developed and investigated.MethodsGNUV205 is an investigational immuno-cytokine molecule which comprises anti-PD-1 antibody (GNUV201) and engineered IL-2 (interleukin-2). Due to GNUV201’s inter-species cross-reactivity to PD-1, GNUV205 can be directly assessed in a murine model without the need for a surrogate antibody. Also, the unique slower dissociation and much stronger binding affinity of GNUV201 to PD-1 in TME (tumor microenvironment)-mimicking low pH condition allows superior selectivity and less side effects of GNUV205. Meanwhile, engineered IL-2 is designed and expected to have minimal adverse effects attributed to its no-alpha (IL-2Rα) and beta/gamma receptor (IL-2Rβγ)-biased engineered structure.ResultsELISA and SPR analysis showed that GNUV205 binds to IL-2Rβγ (CD122/CD132) with attenuated affinity but not to IL-2Rα (CD25). We observed that GNUV205 exhibits enhanced affinity to PD-1 in the low pH condition, a hallmark of the TME. In addition, cell-mediated reporter assay indicated that GNUV205 delivers IL-2 signaling in a PD-1-dependent in-cis manner. When assessed in a MC38 syngeneic in vivo model, multiple- and even a single-dose of GNUV205 were able to exhibit significant anti-tumor efficacy in a dose-dependent manner, with more than 80% tumor clearance at 300 pmole (ca. 2.74 mpk) or higher and without significant effects on body weight change even at 10 x fold higher dose (3,000 pmole). Furthermore, in the tumor re-challenge test, GNUV205 proved to induce tumor-specific long-term immunological memory response as evidenced by 100% tumor rejection even 7 months after the 1st challenge of the cancer cells. We also confirmed GNUV205’s excellent tumor suppressing efficacy in other syngeneic tumor models such as B16F10 and Pan02.ConclusionsIn summary, these data strongly suggest that GNUV205 is a candidate-stage bifunctional immuno-cytokine for solid cancer therapy expected to overcome the limitations of current standard-of-care therapies, such as IL-2 or anti-PD-(L)1s.Ethics ApprovalEvery animal experiment used in this study was reviewed and approved by internal IACUC (E-IACUC2022–002).
Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new ...family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent Km of approximately 22 micromolars, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types
We have identified a homolog of the mammalian ionotropic glutamate receptor genes in Arabidopsis thaliana (AtGluR2). Tbis gene was found to alter Ca2+ utilization when overexpressed in A. thaliana. ...These transgenic plants displayed symptoms of Ca2+ deficiency, including browning and death of the shoot apex, necrosis of leaf tips, and deformation of leaves. Supplementation with Ca2+ alleviated these phenotypes. Overall levels of Ca2+ in tissues of control plants were not significantly different from those of transgenic plants, suggesting that overexpression of the AtGluR2 gene did not affect Ca2+ uptake. However, the relative growth yield as a function of Ca2+ levels revealed that the critical deficiency content of Ca**2+ in transgenic plants was three times higher than that of control plants. The transgenic plants also exhibited hypersensitivity to Na+ and K+ ionic stresses. The ion hypersensitivity was ameliorated by supplementation with Ca2+. The results showed that overexpression of the AtGluR2 gene caused reduced efficiency of Ca2+ utilization in the transgenic plants. The promoter of the AtGluR2 gene was active in vascular tissues, particularly in cells adjacent to the conducting vessels. This suggests that AtGluR2 encodes a functional channel that unloads Ca2+ from the xylem vessels. The results together suggest that appropriate expression of tbe AtGluR2 protein may play critical roles in Ca2+ nutrition by controlling the ion allocation among different Ca2+ sinks both during normal development and during adaptation to ionic stresses.
Summary
Plants develop systemic defense responses upon exposure to pathogens or wounding by herbivores. Lipids and lipid metabolites have previously been implicated in induction of defense molecules ...during plant responses to physical wounding. Possible involvement of changes in lipid composition in systemic wound signal transduction was examined in leaves of seedlings of several different plant species. In the wounded tomato leaf, phosphatidic acid increased approximately fourfold within 5 min whereas lysophosphatidylcholine and lysophosphatidylethanolamine increased over twofold within 15 min of wounding. Similar changes in these lipids were observed in the neighboring non‐wounded leaf. In broad bean, soybean, sunflower and pepper seedlings phosphatidic acid levels increased rapidly and systemically upon wounding. The results suggest that the role of phospholipid hydrolysis and accumulation of lipid metabolites in the early events are responsible for systemic wound signal transduction in plants. Furthermore, they indicate that the wound signal propagates outside the wounded leaf within 5 min in these plants.
As an attempt to examine the transcripts expressed in a single cell type and to unveil the physiology of guard cells at the molecular level, we generated 515 expressed sequence tags (ESTs) from a ...directional cDNA library constructed from guard-cell protoplasts of Brassica campestris L. ssp. pekinensis. A comparative analysis of the guard-cell ESTs against the National Center for Biotechnological Information non-redundant protein database revealed that 133 ESTs (26%) have significant similarity to protein coding sequences in the database. Among them were 35 clones related to genes that have not yet been identified in higher plants. Analysis of RNA gel blots of 14 database-matched clones revealed that five clones harbor the sequences for mRNAs expressed most abundantly in guard cells, one of them detecting an mRNA with highly preferential expression in guard cells. Functional categorization of the putatively identified guard-cell ESTs showed, when compared with maize leaf ESTs, that guard cells expressed a higher proportion of signal transduction components and a lower proportion of structural or photosynthetic genes, as is consistent with the roles of guard cells.
Most cellular processes in an organism depend on functions of expressed sequences. Thus, efficient large-scale functional assignment of expressed sequences is crucial for understanding cellular ...processes. Towards this goal in plants, we designed a "random antisense cDNA mutagenesis (RAM)" approach. In a pilot experiment, 1,000 transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Wassilevskija) expressing random antisense cDNA(s) were generated from Agrobacterium cultures harboring an Arabidopsis antisense cDNA library. We identified 104 mutant lines from the transgenic pool by visual screening. Genetic analysis suggested that 37% of the mutations were likely due to antisense effects. When the cDNA inserts were isolated from 11 mutant lines by polymerase chain reaction and reintroduced into plants to express the antisense transcripts, the original mutant phenotypes were reproduced in 7 cDNA clones. One of the cDNA clones did not generate a database match to any sequence with known functions, but did have a dramatic effect on the architecture of the inflorescence in the antisense transgenic plants. Through the RAM approach, it should be possible to assign a large number of expressed sequences to known in vivo functions in plants.
Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed ...that 21 ESTs show significant similarity to the protein coding sequences in the existing data bases, including five stressor defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach
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