Genome-Wide Identification and Transcriptional Analysis of Arabidopsis DUF506 Gene Family

The Domain of unknown function 506 (DUF506) family, which belongs to the PD-(D/E)XK nuclease superfamily, has not been functionally characterized. In this study, 266 DUF506 domain-containing genes were identified from algae, mosses, and land plants showing their wide occurrence in photosynthetic organisms. Bioinformatics analysis identified 211 high-confidence DUF506 genes across 17 representative land plant species. Phylogenetic modeling classified three groups of plant DUF506 genes that suggested functional preservation among the groups based on conserved gene structure and motifs. Gene duplication and Ka/Ks evolutionary rates revealed that DUF506 genes are under purifying positive selection pressure. Subcellular protein localization analysis revealed that DUF506 proteins were present in different organelles. Transcript analyses showed that 13 of the Arabidopsis DUF506 genes are ubiquitously expressed in various tissues and respond to different abiotic stresses and ABA treatment. Protein-protein interaction network analysis using the STRING-DB, AtPIN (Arabidopsis thaliana Protein Interaction Network), and AI-1 (Arabidopsis Interactome-1) tools indicated that AtDUF506s potentially interact with iron-deficiency response proteins, salt-inducible transcription factors, or calcium sensors (calmodulins), implying that DUF506 genes have distinct biological functions including responses to environmental stimuli, nutrient-deficiencies, and participate in Ca(2+) signaling. Current results provide insightful information regarding the molecular features of the DUF506 family in plants, to support further functional characterizations.

Molecular Characterization of AMPA-Receptor-Containing Vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPA-containing vesicles (ACVs) from whole mouse brains via immunoisolation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography–tandem mass spectrometry (LC–MS/MS). We identified several proteins on ACVs that were previously found to play a role in AMPAR trafficking, including synaptobrevin-2, Rabs, the SM protein Munc18-1, the calcium-sensor synaptotagmin-1, as well as several new candidates, including synaptophysin and synaptogyrin on ACV membranes. Additionally, we identified two populations of ACVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ACVs, and larger transferrin- receptor-, GluA1-, GluA2-, and GluA3-containing ACVs. The small-diameter population of ACVs may represent a fusion-capable population of vesicles due to the presence of synaptobrevin-2. Because the fusion of ACVs may be a requisite of LTP, this population could represent trafficking vesicles related to LTP.

Rational engineering of ratiometric calcium sensors with bright green and red fluorescent proteins

Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. Recently developed ratiometric GECIs primarily employ cyan and yellow-fluorescent fluorescence resonance energy transfer pairs, and thus fall short in some applications that require deep tissue penetration and resistance to photobleaching. We engineered a set of green-red ratiometric calcium sensors that fused two fluorescent proteins and calcium sensing domain within an alternate configuration. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins. These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. In turn, the shot-noise limited signal fidelity of our sensors when reporting action potentials in cultured neurons and in the awake behaving mice was higher than the fidelity of existing sensors. Our sensor enabled a regime of imaging that simultaneously captured neural structure and function down to the deep layers of the mouse cortex.

Molecular characterization of AMPA receptor trafficking vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPAR trafficking vesicles (ATVs) from whole mouse brains via immunoprecipitation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified several proteins on ATVs that were previously found to play a role in AMPAR trafficking, including SNARES (including synaptobrevin 2), Rabs, the SM protein Munc18-1, a calcium-sensor (synaptotagmin-1), as well as several new markers, including synaptophysin and synaptogyrin on ATV membranes. Additionally, we identified two populations of ATVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ATVs and larger transferrin-receptor-, GluA1-, GluA2-, GluA3-containing ATVs. The smaller population of ATVs likely represents a trafficking vesicle whose fusion is essential for LTP. These findings reveal the important role of AMPAR sorting into fusion-competent trafficking vesicles that are implicated in synaptic strength modification and reveal candidates of putative effectors and regulators of AMPAR trafficking.

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

Impairment of Store-operated Calcium Entry: Implications in Alzheimer’s Neurodegeneration

Alzheimer's disease (AD) is an insidious and progressive neurodegenerative disorder. Dysfunction of central cholinergic neurons, amyloid aggregation and deposition,oxidative stress,and biometal dyshomeostasis has been regarded as the major pathogenic mediators in this devastating disease. However, strategies derived from these hypotheses fail to slow down or stop the progression of AD, warranting a combination of therapies to target multiple etiological factors or examining alternative hypothesis. Store-operated calcium entry (SOCE) is the process by which depletion of calcium in the endoplasmic reticulum (ER) lumen causes an influx of calcium across plasmalemma. Accumulating evidence indicates that neuronal SOCE (nSOCE) is inhibited in family AD (FAD) and the inhibition of which causes instability of dendritic spines and enhances amyloidogenesis. Mutant Presenilin fails to function as an ER calcium leak channel and promotes degradation of stromal interaction molecules (STIM), ER calcium sensors; these effects may account for the repression of nSOCE in FAD. We have demonstrated that activation of autophagy degrades STIM proteins, resulting in a trimming effect on a dendritic arbor, under proteasome inhibition and endoplasmic reticulum stress, which are intimately connected with AD. Thus, we hypothesize that autophagy represses SOCE by degrading STIM proteins, leading to synapse loss in AD. This review article will highlight the roles of SOCE in AD neurodegeneration, the degradative mechanisms of STIM protein, and the therapeutic potential and associated challenge.

Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release

ABSTRACTDopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6‐ hydroxydopamine model of Parkinson’s disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

Calcium Sensors STIM1 and STIM2 Regulate Different Calcium Functions in Cultured Hippocampal Neurons

There are growing indications for the involvement of calcium stores in the plastic properties of neurons and particularly in dendritic spines of central neurons. The store-operated calcium entry (SOCE) channels are assumed to be activated by the calcium sensor stromal interaction molecule (STIM)which leads to activation of its associated Orai channel. There are two STIM species, and the differential role of the two in SOCE is not entirely clear. In the present study, we were able to distinguish between transfected STIM1, which is more mobile primarily in young neurons, and STIM2 which is less mobile and more prominent in older neurons in culture. STIM1 mobility is associated with spontaneous calcium sparks, local transient rise in cytosolic [Ca2+]i, and in the formation and elongation of dendritic filopodia/spines. In contrast, STIM2 is associated with older neurons, where it is mobile and moves into dendritic spines primarily when cytosolic [Ca2+]i levels are reduced, apparently to activate resident Orai channels. These results highlight a role for STIM1 in the regulation of [Ca2+]i fluctuations associated with the formation of dendritic spines or filopodia in the developing neuron, whereas STIM2 is associated with the maintenance of calcium entry into stores in the adult neuron.

All-Solid-State Calcium Sensors Modified with Polypyrrol (PPY) and Graphene Oxide (GO) as Solid-Contact Ion-to-Electron Transducers

Reliable, cost-effective, and robust screen-printed sensors were constructed and presented for Ca2+ ions determination. The sensors were based on the use of bilirubin (1,3,6,7-tetramethyl-4,5- dicarboxyethy-2,8-divinyl-(b-13)-dihydrobilenone) as a recognition sensory material in plasticized poly (vinyl chloride) (PVC) membranes. Polypyrrol (PPY) and graphene oxide (GO) were used as ion-to-electron transducers, where the effects of anionic excluder, pH, and selectivity were investigated. In a 50 mM tris buffer solution of pH 5, the electrodes offered a potential response for Ca2+ ions with a near-Nernstian slopes of 38.1 ± 0.4 (r2 = 0.996) and 31.1 ± 0.6 (r2 = 0.999), detection limits 3.8 × 10−6 (0.152 μg/mL) and 2.3 × 10−7 M (8.0 ng/mL), and linear concentration ranges of 7.0 × 10−6–1.0 × 10−2 (400–0.28 μg/mL) and 7.0 × 10−7–1.0 × 10−2 M (400–0.028 μg/mL) for sensors based on PPY and GO, respectively. Both sensors revealed stable potentiometric responses with excellent reproducibility and enhanced selectivity over a number of most common metal ions, such as Na+, K+, Li+, NH4+, Fe2+, Mg2+, and Ba2+. Impedance spectroscopy and chronopotentiometric techniques were used for evaluating the potential drift and the interfacial sensor capacitance. The proposed sensors offered the advantages of simple design, ability of miniaturization, good potential stability, and cost-effectiveness. The developed electrodes were applied successfully to Ca2+ ion assessment in different pharmaceutical products, baby-food formulations, and human blood samples. The results obtained were compared with data obtained by atomic absorption spectrometry (AAS).