Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.Fil: Aprile García, Fernando. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Metzger, Michael W.. Max Planck Institute of Psychiatry; AlemaniaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Stadler, Herbert. Affectis Pharmaceuticals; AlemaniaFil: Acuña, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hoijman, Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Refojo, Damian. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mitkovski, Mišo. Max Planck Institute of Experimental Medicine; AlemaniaFil: Panhuysen, Markus. Affectis Pharmaceuticals; AlemaniaFil: Stühmer, Walter. Max Planck Institute of Experimental Medicine; AlemaniaFil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania. HMNC Brain Health; AlemaniaFil: Deussing, Jan M.. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images

Methods of blind source separation are used in many contexts to separate composite data sets according to their sources. Multiply labeled fluorescence microscopy images represent such sets, in which the sources are the individual labels. Their distributions are the quantities of interest and have to be extracted from the images. This is often challenging, since the recorded emission spectra of fluorescent dyes are environment- and instrument-specific. We have developed a nonnegative matrix factorization (NMF) algorithm to detect and separate spectrally distinct components of multiply labeled fluorescence images. It operates on spectrally resolved images and delivers both the emission spectra of the identified components and images of their abundance. We tested the proposed method using biological samples labeled with up to four spectrally overlapping fluorescent labels. In most cases, NMF accurately decomposed the images into contributions of individual dyes. However, the solutions are not unique when spectra overlap strongly or when images are diffuse in their structure. To arrive at satisfactory results in such cases, we extended NMF to incorporate preexisting qualitative knowledge about spectra and label distributions. We show how data acquired through excitations at two or three different wavelengths can be integrated and that multiple excitations greatly facilitate the decomposition. By allowing reliable decomposition in cases where the spectra of the individual labels are not known or are known only inaccurately, the proposed algorithms greatly extend the range of questions that can be addressed with quantitative microscopy

The SocioBox: A Novel Paradigm to Assess Complex Social Recognition in Male Mice.

Impairments in social skills are central to mental disease, and developing tools for their assessment in mouse models is essential. Here we present the SocioBox, a new behavioral paradigm to measure social recognition. Using this paradigm, we show that male wildtype mice of different strains can readily identify an unfamiliar mouse among 5 newly acquainted animals. In contrast, female mice exhibit lower locomotor activity during social exploration in the SocioBox compared to males and do not seem to discriminate between acquainted and unfamiliar mice, likely reflecting inherent differences in gender-specific territorial tasks. In addition to a simple quantification of social interaction time of mice grounded on predefined spatial zones (zone-based method), we developed a set of unbiased, data-driven analysis tools based on heat map representations and characterized by greater sensitivity. First proof-of-principle that the SocioBox allows diagnosis of social recognition deficits is provided using male PSD-95 heterozygous knockout mice, a mouse model related to psychiatric pathophysiology.peerReviewe

hP2X7R-Gln460Arg interacts with hP2X7R-WT at the cell membrane.

<p>(A) Immunoprecipitation (IP) assays on HEK293 clones constitutively co-expressing STREP-tagged hP2X7R-WT and HIS-tagged hP2X7R-Gln460Arg, using anti-HIS (αHIS) and anti-STREP (αSTREP) antibodies for the immunoblotting (IB). One representative experiment out of four with similar results is shown from two out of four clones analyzed. Mock: IP performed in parallel with normal mouse IgGs. Right: Control panel showing that neither HIS-tagged hP2X7R-Gln460Arg nor STREP-tagged hP2X7R-WT were detected in untransfected HEK293 cells. Duplicates are shown. (B) FRET-based confirmation of interaction between hP2X7R-Gln460Arg with hP2X7R-WT. Pixel-by-pixel quantification of sensitized emission FRET on living cells. First column: FRET image: Ex 458 nm/Em 530–630 nm. Second and third columns: Cerulean and cp49Venus fluorescence. The fourth column displays the NFRET image of the same cell. Brighter pixels show higher NFRET levels. Pixels with signal amplitude below threshold are shaded blue. Representative cell images for each condition are shown. Insets: representative magnifications of membrane areas where quantifications were performed. FRET Quantification: Measurement of FRET levels in the cell membrane. Each bar represents the mean ± s.e.m. of 5–10 cells, delimiting 4–6 ROIs per cell at the membrane level, in four independent experiments.</p

Co-expression of hP2X7R-Gln460Arg with hP2X7R-WT diminishes normal receptor function.

<p>(A) Increase of intracellular calcium of stably transfected HEK293 cells was measured following BzATP application (50 μM) (repeated measures ANOVA, <i>P</i> < 0.01 hP2X7R-WT and hP2X7R-Gln460Arg versus HEK293; n = 4). For each cell line, nine individual clones were analyzed (B) Left: representative whole-cell measurements out of four independent experiments by whole-cell patch clamp analysis. Right: Quantification of inward currents elicited by BzATP (One-way ANOVA with Scheffé’s test, ns = non-significant versus hP2X7R-WT; n = 4) (C) Increase of intracellular calcium of HEK293 cells stably transfected with hP2X7R-WT (9 clones) and stably double transfected with hP2X7R-WT + hP2X7R-Gln460Arg (10 clones) was measured (repeated measures ANOVA, <i>P</i> < 0.01 hP2X7R-WT + Gln460Arg versus hP2X7R-WT; n = 4). (D) Left: representative whole-cell measurements by whole-cell patch clamp analysis. Right: Quantification of inward currents elicited by BzATP (One-way ANOVA with Scheffé’s test, *<i>P</i> < 0.05 versus hP2X7R-WT; n = 4) (E) BzATP (50 μM)-induced activation of p-ERK 1/2 in HEK293 cells expressing P2X7R variants. Each value of pERK1/2 was normalized to total ERK1/2. Results are expressed as the percentage of maximum pERK1/2 obtained at 2 minutes of stimulation in hP2X7R-WT cells ± s.e.m. from 3 independent experiments. One-way ANOVA, * P < 0.05 versus hP2X7R-WT and versus hP2X7R-Gln460Arg at the same time points. Bottom panels show WBs of pERK1/2 and total ERK1/2 from a representative experiment. (+): Fetal calf serum 10% treatment for 10 min, positive control for p-ERK 1/2 activation. Inset: Quantification and representative example showing WB detection of hP2X7R variants in parental HEK293 cells and analyzed stable clones.</p

Silencing of either subunit in cells co-expressing hP2X7R-Gln460Arg and hP2X7R-WT restores normal P2X7R function.

<p>(A) Increase of intracellular calcium triggered by BzATP (50 μM) was evaluated in hP2X7R-WT and hP2X7R-Gln460Arg co-expressing HEK293 cells transfected with the corresponding scramble siRNA control, WT- or Gln460Arg-specific P2X7R siRNAs (100 nM) for 72 h (repeated measures ANOVA, <i>P</i> < 0.001 siRNA hP2X7R-WT and siRNA hP2X7R-Gln460Arg versus scramble siRNA; n = 4). (B) Silencing of mRNA coding for P2X7R-WT or P2X7R-Gln460Arg was confirmed by quantitative real-time RT-PCR (One-way ANOVA, *<i>P</i> < 0.01 versus scramble siRNA; n = 3). (C) BzATP (50 μM)-induced activation of p-ERK 1/2 in HEK293 cells expressing hP2X7R-WT (left), hP2X7R-Gln460Arg (middle) and co-expressing hP2X7R-WT and hP2X7R-Gln460Arg (right). Each value of pERK1/2 was normalized to total ERK1/2. Results are expressed as the percentage of pERK1/2 obtained at 6 minutes of stimulation in hP2X7R-WT cells ± s.e.m. from 3 independent experiments. One-way ANOVA, * P < 0.05 versus hP2X7R-WT and versus hP2X7R-Gln460Arg at the same time points. WBs from a representative experiment are shown. (D) HEK293 cells co-expressing hP2X7R-WT and hP2X7R-Gln460Arg P2X7R variants were silenced using siRNAs that specifically target hP2X7R-Gln460Arg (left), hP2X7R-WT (middle) and with the corresponding scramble siRNA as a control (right). Each value of pERK1/2 was normalized to total ERK1/2. Results are expressed as the percentage of pERK1/2 obtained at 6 minutes of stimulation in siRNA hP2X7R-Gln460Arg cells ± s.e.m. from 3 independent experiments. One-way ANOVA, * P < 0.05 versus siRNA hP2X7R-WT and versus siRNA hP2X7R-Gln460Arg at the same time points. WBs from a representative experiment are shown.</p

Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.

Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes