Resting-state gamma-band power alterations in schizophrenia reveal E/I-balance abnormalities across illness-stages

We examined alterations in E/I-balance in schizophrenia (ScZ) through measurements of resting-state gamma-band activity in participants meeting clinical high-risk (CHR) criteria (n = 88), 21 first episode (FEP) patients and 34 chronic ScZ-patients. Furthermore, MRS-data were obtained in CHR-participants and matched controls. Magnetoencephalographic (MEG) resting-state activity was examined at source level and MEG-data were correlated with neuropsychological scores and clinical symptoms. CHR-participants were characterized by increased 64–90 Hz power. In contrast, FEP- and ScZ-patients showed aberrant spectral power at both low- and high gamma-band frequencies. MRS-data showed a shift in E/I-balance toward increased excitation in CHR-participants, which correlated with increased occipital gamma-band power. Finally, neuropsychological deficits and clinical symptoms in FEP and ScZ-patients were correlated with reduced gamma band-activity, while elevated psychotic symptoms in the CHR group showed the opposite relationship. The current study suggests that resting-state gamma-band power and altered Glx/GABA ratio indicate changes in E/I-balance parameters across illness stages in ScZ

The C Isoform of <i>Dictyostelium</i> Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress

<div><p>Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from <i>Dictyostelium discoideum</i>, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for <i>TspA-E</i> in the multicellular slug stage, whereas vegetative cells expressed only <i>TspA</i>, <i>TspC</i> and, to a lesser extent, <i>TspD</i>. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. <i>N</i>-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC <i>in vivo</i>. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of <i>TspC</i> and <i>TspD</i> exibited unaltered growth, adhesion, random motility and development. Yet, <i>tspC</i>^<i>−</i> cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of <i>TspC</i> rescued their phenotype. In conclusion, our data fill a gap in <i>Dictyostelium</i> research and open up the possibility that Tsps in contractile vacuoles of e.g. <i>Trypanosoma</i> may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.</p></div

Schematic representation of human and <i>D</i>. <i>discoideum</i> Tsp topologies.

<p>A generic topology of human Tsp is shown on the left (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref033" target="_blank">33</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref005" target="_blank">5</a>]), the proposed <i>Dictyostelium</i> Tsp topology on the right, as inferred from protein structure predictions and protein sequence alignments. Numbers in blue indicate the range of amino acids (aa) in the N-terminus, the small (EC1) and large (EC2) extracellular loops, the inner cytoplasmic loop (ICL) and the C-terminus. Blue and yellow shadings represent the variable (protein-protein interactions) and conserved domain of EC2, respectively. Cysteines in yellow are 100% conserved, red circles represent the “CCG”-motif that is altered (X) in four of five <i>D</i>. <i>discoideum</i> Tsps. Conserved residues in the transmembrane regions are indicated in italic letters. Potential disulfide bridges are represented by blue lines. Palmitoylation sites as predicted by CSS Palm 1.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref058" target="_blank">58</a>] are marked by pink wavy lines. Black pins show potential <i>N</i>-glycosylation sites (N x T/S). Please note that not all Tsps may undergo these modifications to the same extent.</p

Twenty-four hour development.

<p>Cells were starved on KK2 agar plates and incubated at 22°C in a moist chamber. Pictures were taken at the indicated time points illustrating aggregation (4 h), streaming (8 h), culmination (16 h) and spore-head formation (24 h). Development of <i>tspC</i>^<i>−</i>, as well as of <i>tspD</i>^<i>−</i> mutant was unaltered compared to wildtype cells. Scale bars = 100 μm.</p

Expression of <i>TspA-E</i> in <i>D</i>. <i>discoideum</i>.

<p>Total RNA was isolated from growth phase cells (amoebae) and from cells after 16 h of development (slugs), cDNA was prepared and PCR reactions were performed with sequence-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.s003" target="_blank">S1 Table</a>). PCR products had the predicted sizes of approximately 700 bp.</p

Generation of TspA, TspC and TspD antibodies, Western blots of Tsp-GFP fusion proteins and specification of the TspA antibody.

<p>(A) Partial sequence alignment of <i>D</i>. <i>discoideum</i> TspA-E and human CD9 (the plot was generated using TEXshade [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref059" target="_blank">59</a>]). Peptide sequences that were used for antibody production and conserved Cys residues are labeled in white. The conserved “CCG”-motif is indicated. (B) Western blots of cell lysates (30 μg protein per lane) of <i>D</i>. <i>discoideum</i> vegetative cells transformed with the control plasmid pDM323 (−) or <i>Tsp-GFP</i> using a polyclonal α-GFP antibody as well as the affinity purified antibodies α-TspA and α-TspC. The specificity of the anti-TspA antiserum (preblock) was tested by a 1 h preincubation with 1 μg ml^-1 of the immunizing peptide corresponding to a 10-fold excess. (C) Western blot of cell lysates (88 μg protein per lane) of <i>D</i>. <i>discoideum</i> vegetative cells (−) and of cells transformed with <i>TspA-His</i> using the affinity purified α-TspA antibody as well as a monoclonal α-His antibody. * = unspecific band.</p

Overview of Tsps in <i>D</i>. <i>discoideum</i>.

<p>Overview of Tsps in <i>D</i>. <i>discoideum</i>.</p

Tsp-GFP fusion constructs co-localize with the proton pump on contractile vacuoles.

<p>(A) Fixed amoebae expressing TspC-GFP show a delicate green network with occasional bladders at the bottom of the cell that does not co-localize with a marker of the ER (PDI, red) in indirect immunofluorescence. OL = overlay. (B) In a more medial plane of the same cell, the GFP signal is found within the plasma membrane in addition to having a cloudy appearance (arrowheads). This is indicative of the Golgi-apparatus lying close to the ER-rings that represent the nuclear envelopes. (C) Using the V-H⁺ ATPase (or proton pump, PP, red) as a stain for the CV, the cisternae (arrows) and tubular elements (arrowheads) strongly overlap with TspC-GFP in the overlay (OL). Some vesicular structures only labeled by the V-H⁺ ATPase antibody may be components of the endolysosomal system that is known to be populated by the V-H⁺ ATPases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162065#pone.0162065.ref060" target="_blank">60</a>]. N-terminal (D–F) as well as C-terminal (G–H) GFP fusions to TspA, TspC and TspD proteins all localize to bladders and tubules of the CV system (merge yellow) counterstained as in (C). Scale bars = 5 μm.</p

Disruption of the <i>TspC</i> and <i>TspD</i> genes.

<p>(A) Genomic 5´ and 3´ fragments of <i>TspC</i> and <i>TspD</i> were amplified by PCR, ligated into a blasticidin-resistance cassette and each construct was transferred into AX2 wildtype cells by electroporation. The adjacent genes of <i>TspC</i> (<i>lipocalin</i>, DDB_G0269882; <i>pseudogene samkB</i>, DDB_G0270988) and <i>TspD</i> (<i>TspA</i>; DDB_G0269884) are shown. (B) For each gene, two independent blasticidin resistant clones were compared with the wildtype (wt) for correct genomic 5´ integration (primers 1/2), insertion of the blasticidin-resistance gene (primers 3/4), and 3´ integration (primers 5/6).</p