Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4

Praktyka kliniczna oceny minimalnej choroby resztkowej u chorych na szpiczaka plazmocytowego w Polsce: badanie ankietowe Polskiego Konsorcjum Szpiczakowego

Studies exploring the significance of minimal residual disease (MRD) in plasma cell myeloma (PCM) have proven its prognostic value, regardless of the type of administered treatment. In order to assess the current practice for evaluating MRD in Poland, we conducted a survey on the methods for assessing MRD and on the MRD testing time points at Polish hematological centres. Seven out of 15 institutions surveyed use of the flow cytometry (FC) method for MRD assessment. The FC-MRD assessment is performed uniformly only in those patients achieving complete remission(CR). However, the specific indications and assessment time points differed at the tested centres including: testing MRD only after autologous hematopoietic stem cell transplantation (auto-HSCT), after auto-HSCT and consolidation, after completion of first line chemotherapy or after obtaining CR in any line of treatment. The study also showed considerable heterogeneity in the FC-MRD methodology, which affects test sensitivity (from 10–3 to 10–5). None of the surveyed centres uses molecular techniques for MRD assessment. In 8 of the 15 institutions, patients are monitored by imaging techniques. Our survey may thus be useful for developing guidelines and standardization of MRD assessment in PCM in Poland.W badaniach nad znaczeniem minimalnej choroby resztkowej (MRD) w szpiczaku plazmocytowym (PCM) dowiedziono, że status MRD ma wartość prognostyczną niezależnie od zastosowanego leczenia. W celu poznania zasad monitorowania MRD u chorych na PCM w polskich ośrodkach hematologicznych przeprowadzono badanie ankietowe. W ankiecie zadano pytania dotyczące stosowanych metod wykrywania MRD oraz punktów czasowych, w których badania są wykonywane. W 7 z 15 ośrodków objętych badaniem ankietowym oznaczenia MRD w PCM wykonuje się w aspiratach szpiku kostnego metodą cytometrii przepływowej (FC). We wszystkich ośrodkach oznaczenia FC-MRD są wykonywane jedynie u chorych w całkowitej remisji (CR), jednak w różnych punktach czasowych — tylko po autologicznym przeszczepieniu krwiotwórczych komórek macierzystych (allo-HSCT), po allo-HSCT i konsolidacji, po zakończeniu leczenia pierwszej linii lub, w przypadku uzyskania CR, po dowolnej linii leczenia. Stwierdzono ponadto znaczne różnice w sposobie wykonywania badania FC-MRD wpływające na osiąganą czułość detekcji MRD (od 10–3 do 10–5). W żadnym z ankietowanych ośrodków nie ocenia się MRD w szpiku kostnym technikami molekularnymi. Monitorowanie choroby resztkowej metodami obrazowymi stosuje personel 8 z 15 ośrodków. Wyniki przeprowadzonej ankiety mogą posłużyć wypracowaniu wspólnych wytycznych i standaryzacji oceny MRD w PCM w Polsce

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability

Deletion of RA domain in SNX27b impairs functional regulation of GIRK channels.

<p><b>A</b>, Cartoon shows model of GIRK channels regulation by SNX27b. GIRK channels recycle through endosomal compartments. SNX27b associating with GIRK2c/3 channels in the early endosome (EE) reduces plasma membrane expression (PM) by targeting some channels to the late endosome (LE). <b>B</b>, SNX27 contains three functional domains; PDZ, PX and RA. GIRK2c and GIRK3 contain a C-terminal PDZ binding motif (-E(S/N)ESKV). <b>C,</b> Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293T cells transfected with cDNA for GABA_B1a/B2 receptors, GIRK2c/GIRK3 and either control vector, SNX27b or SNX27b-ΔRA. Agonist-independent basal currents are revealed by inhibition with 1 mM Ba²⁺. <b>D</b>, Average baclofen-induced current densities (I_Baclofen) for control (–41.3±5.2 pA⋅pF⁻¹, n = 8), SNX27b (–11.0±3.6 pA⋅pF⁻¹, n = 8) and SNX27b- ΔRA (–55.9±8.2 pA⋅pF⁻¹, n = 13) with GIRK2c/GIRK3. <b>E</b>, Average I_Baclofen for control (–15.7±3.6 pA⋅pF⁻¹, n = 6) and SNX27b-ΔRA (–18.6±4.9 pA⋅pF⁻¹, n = 5) with GIRK1/GIRK3 (**P<0.05, one way ANOVA followed by Bonferroni <i>post hoc</i> test; n.s. – not significant).</p

Dominant-negative H-Ras (H-Ras_S17NDN) prevents SNX27b-dependent down-regulation of GIRK channels.

<p><b>A</b>, Schematic shows GIRK2c and GIRK3 with PDZ-binding motif and GIRK2a and GIRK3-RR, which lack motifs that interact with SNX27-PDZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Balana1" target="_blank">[7]</a>. <b>B</b>, Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293 cells transfected with cDNA for GABA_B1a/B2, GIRK2c/GIRK3 and either empty vector (Control), H-Ras_S17NDN or H-Ras_S17NDN plus SNX27b. <b>C</b>, Bar graph shows average I_Baclofen for GIRK2c/GIRK3 alone (–47.7±8.5 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–22.0±3.8 pA⋅pF⁻¹, n = 19) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–23.7±4.7 pA⋅pF⁻¹, n = 16). <b>D</b>, Bar graph shows I_Baclofen for control (GIRK2a/GIRK3-RR alone) (–18.8±3.0 pA⋅pF⁻¹, n = 10) and GIRK2a/GIRK3-RR plus H-Ras_S17NDN (–18.6±2.0 pA⋅pF⁻¹, n = 11). <b>E</b>, Bar graph shows average I_Barium for GIRK2c/GIRK3 alone (–27.6±9.8 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–7.3±2.2 pA⋅pF⁻¹, n = 18) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–7.1±1.5 pA⋅pF⁻¹, n = 14). <b>F</b>, Bar graph shows average I_Barium for GIRK2a/GIRK3-RR alone (−2.49±0.9 pA⋅pF⁻¹, n = 10), GIRK2c/GIRK3 and H-Ras_S17NDN (−1.84±0.4 pA⋅pF⁻¹, n = 11). **P<0.05, one way ANOVA followed by Bonferroni post hoc test; n.s. – not significant.</p

Deletion of RA domain in SNX27b affects localization of GIRK2c/3 channels monitored with BiFC.

<p><b>A</b>, <i>Left</i>, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. <i>Right</i>, BiFC-tagged GIRK2c/3 channels are functional. Current-voltage plot is shown for ^CYGIRK2c/^NYGIRK3 channels. Baclofen (100 µM) activates and Ba²⁺ (1 mM) inhibits inwardly rectifying current. HEK293T cells were transfected with cDNA encoding GABA_B1a, GABA_B2 and ^CYGIRK2c/^NYGIRK3. Average baclofen-induced current densities were –13.2±6.0 pA⋅pF⁻¹ (n = 3) for ^CYGIRK2c/^NYGIRK3. <b>B</b>, HEK293 cells were co-transfected with ^CYGIRK2c, ^NYGIRK3 and either control cDNA (<i>i</i>), wild-type SNX27b (<i>ii</i>), SNX27b-ΔRA (deletion of Asp272-Trp358) (<i>iii</i>) or SNX27b-Y51L (a PDZ mutation) (<i>iv</i>). Green fluorescence in images represents molecular recombination of ^CYGIRK2c/^NYGIRK3 heterotetramers. Coexpression of wild-type SNX27b induced formation of puncta. By contrast, ^CYGIRK2c/^NYGIRK3 fluorescence was diffuse in the cytoplasm for SNX27b-ΔRA and for SNX27b-Y51L, similar to control. Inset shows zoom of boxed area. <b>C,</b> SNX27b-ΔRA exhibited a pattern of punctate expression similar that of wild-type SNX27b. YFP was fused to the C-terminus of SNX27b or SNX27b-ΔRA to directly visualize expression. HEK293T cells were transfected with cDNA for SNX27b-YFP and SNX27bΔRA-YFP. Scale bar: 10 µm.</p