Hydroxynorketamine, but not ketamine, acts via α7 nicotinic acetylcholine receptor to control presynaptic function and gene expression.

Ketamine is clinically used fast-acting antidepressant. Its metabolite hydroxynorketamine (HNK) shows a robust antidepressant effect in animal studies. It is unclear, how these chemically distinct compounds converge on similar neuronal effects. While KET acts mostly as N-methyl-d-aspartate receptor (NMDAR) antagonist, the molecular target of HNK remains enigmatic. Here, we show that KET and HNK converge on rapid inhibition of glutamate release by reducing the release competence of synaptic vesicles and induce nuclear translocation of pCREB that controls expression of neuroplasticity genes connected to KET- and HNK-mediated antidepressant action. Ro25-6981, a selective antagonist of GluN2B, mimics effect of KET indicating that GluN2B-containing NMDAR might mediate the presynaptic effect of KET. Selective antagonist of α7 nicotinic acetylcholine receptors (α7nAChRs) or genetic deletion of Chrna7, its pore-forming subunit, fully abolishes HNK-induced synaptic and nuclear regulations, but leaves KET-dependent cellular effects unaffected. Thus, KET or HNK-induced modulation of synaptic transmission and nuclear translocation of pCREB can be mediated by selective signaling via NMDAR or α7nAChRs, respectively. Due to the rapid metabolism of KET to HNK, it is conceivable that subsequent modulation of glutamatergic and cholinergic neurotransmission affects circuits in a cell-type-specific manner and contributes to the therapeutic potency of KET. This finding promotes further exploration of new combined medications for mood disorders.

miR96- and miR182-driven regulation of cytoskeleton results in inhibition of glioblastoma motility.

Glioblastoma multiforme (GBM) is one of the most common forms of brain tumor. As an excessively invasive tumor type, GBM cannot be fully cured due to its invasion ability into healthy brain tissues. Therefore, molecular mechanisms behind GBM migration and invasion need to be deeply investigated for the development of effective GBM treatments. Cellular motility and invasion are strictly associated with the cytoskeleton, especially with actins and tubulins. Palladin, an actin-binding protein, tightly bundles actins during initial invadopodia and contraction fiber formations, which are essential for cellular motility. Spastin, a microtubule-binding protein, cuts microtubules into small pieces and acts on invadopodia elongation and cellular trafficking of invadopodia-associated proteins. Regulation of proteins such as spastin and palladin involved in dynamic reorganization of the cytoskeleton, are rapidly carried out by microRNAs at the posttranscriptional level. Therefore, determining possible regulatory miRNAs of spastin and palladin is critical to elucidate GBM motility. miR96 and miR182 down-regulate SPAST and PALLD at both transcript and protein levels. Over-expression of miR96 and miR182 resulted in inhibition of the motility. However, over-expression of spastin and palladin induced the motility. Spastin and palladin rescue of miR96- or miR182-transfected U251 MG cells resulted in diminished effects of the miRNAs and rescued the motility. Our results demonstrate that miR96 and miR182 over-expressions inhibit GBM motility by regulating cytoskeleton through spastin and palladin. These findings suggest that miR96 and miR182 should be investigated in more detail for their potential use in GBM therapy.

CRISPR/Cas9-mediated generation of hESC lines with homozygote and heterozygote p.R331W mutation in CTBP1 to model HADDTS syndrome.

C-terminal Binding Protein 1 (CTBP1) is a ubiquitously expressed transcriptional co-repressor and membrane trafficking regulator. A recurrent de novo c.991C>T mutation in CTBP1 leads to expression of p.R331W CTBP1 and causes hypotonia, ataxia, developmental delay, and tooth enamel defects syndrome (HADDTS), a rare early onset neurodevelopmental disorder. We generated hESCs lines with heterozygote and homozygote c.991C>T in CTBP1 using CRISPR/Cas9 genome editing and validated them for genetic integrity, off-target mutations, and pluripotency. They will be useful for investigation of HADDTS pathophysiology and for screening for potential therapeutics.

Development and Application of Automatized Routines for Optical Analysis of Synaptic Activity Evoked by Chemical and Electrical Stimulation.

The recent development of cellular imaging techniques and the application of genetically encoded sensors of neuronal activity led to significant methodological progress in neurobiological studies. These methods often result in complex and large data sets consisting of image stacks or sets of multichannel fluorescent images. The detection of synapses, visualized by fluorescence labeling, is one major challenge in the analysis of these datasets, due to variations in synapse shape, size, and fluorescence intensity across the images. For their detection, most labs use manual or semi-manual techniques that are time-consuming and error-prone. We developed SynEdgeWs, a MATLAB-based segmentation algorithm that combines the application of an edge filter, morphological operators, and marker-controlled watershed segmentation. SynEdgeWs does not need training data and works with low user intervention. It was superior to methods based on cutoff thresholds and local maximum guided approaches in a realistic set of data. We implemented SynEdgeWs in two automatized routines that allow accurate, direct, and unbiased identification of fluorescently labeled synaptic puncta and their consecutive analysis. SynEval routine enables the analysis of three-channel images, and ImgSegRout routine processes image stacks. We tested the feasibility of ImgSegRout on a realistic live-cell imaging data set from experiments designed to monitor neurotransmitter release using synaptic phluorins. Finally, we applied SynEval to compare synaptic vesicle recycling evoked by electrical field stimulation and chemical depolarization in dissociated cortical cultures. Our data indicate that while the proportion of active synapses does not differ between stimulation modes, significantly more vesicles are mobilized upon chemical depolarization.

Tissue-specific transcriptional regulation of seven heavy metal stress-responsive miRNAs and their putative targets in nickel indicator castor bean (R. communis L.) plants.

R.communis L. has high capability to accumulate nickel which is a trace nutrient for higher plants and also an environmental contaminant causes toxicity related symptoms at higher concentrations. MicroRNAs (miRNAs) are known to be important modulators of responses against heavy metal stress for detoxification of the metal. In this study, we experimentally measured and validated the transcript levels of the seven heavy metal stress response-related miRNAs and the expression levels of target genes in both leaf and root tissues of R. communis L. subjected to three different concentrations of nickel stress via qRT-PCR quantification. The results demonstrated differential regulations of heavy metal stress-responsive miRNAs and their putative targets in both tissues in same stress treatments. This dynamic regulation suggest that regulatory processes differ between the tissues under nickel stress. Our data suggest that, miR838 was the most responsive to the Ni2+ stress. miR398 target gene Cu-Zn/SOD was found to be up-regulated in both root and leaf tissues. The relations between TCP and expression levels of miR159 and miR319 were also found statistically significant exclusive to leaf tissues. In leaf tissue, changes in miR395 level and its putative target genes, sulphate transporter and sulphate adenyltransferase gene were found in relation whereas, only expression level of sulphate transporter represented a statistically significant relation in root tissue. The sharp decrease in transcript levels of 2r3 myb gene at lower nickel dose suggest to investigate the role of r2r3 myb and the all MYB family members in primary and secondary metabolisms against nickel stress.

Transcript profiling of salt tolerant tobacco mutants generated via mutation breeding.

The main aim of the study is to identify the genes differentially, predominantly or specifically expressed in salt tolerant tobacco mutants, improved from Akhisar 97 and Izmir Özbas varieties via mutation breeding, with respect to unstressed control plants. Seven tobacco mutants which have different salt tolerance capacities were evaluated by Gene Fishing analysis. Under stress conditions differentially expressed 100 reproducible bands were identified (74 of up-regulated and 20 of down-regulated while 6 were unknown). 75 of differentially expressed genes (DEGs) were successfully extracted from the gel and sequence analyses were performed. Functional annotation of the DEGs was performed against Blastn by interrogating their sequences. The 65 salt-regulated differentially expressed genes showed similarity with known genes, while 6 of DEGs didn't show any genetic similarities with known genes. DEGs were classified in eleven functional categories involving the abiotic stress response, biotic stress response, energy metabolism, cellular transport, catalitic activity, protein modification, amino acid metabolism and transcription factors. All the mutants were evaluated for their regulatory mechanisms against salt stress. The current data reveal that these six DEGs should be identified by next generation sequencing techniques and functional analysis should be design to understand the role of these six differentially expressed genes of tobacco mutants in further studies to improve new genetic resources.