Subcellular specificity of cannabinoid effects in striatonigral circuits

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior

Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of <i>Drosophila melanogaster</i>: Implication in mitochondrial homeostasis

<div><p>The small heat shock protein (sHsp) Hsp22 from <i>Drosophila melanogaster</i> (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient <i>in vitro</i> chaperone, its function within mitochondria <i>in vivo</i> remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.</p></div

Gene Ontology analysis of DmHsp22-associated proteins.

<p>(A) The potential partners of DmHsp22 were regrouped in 3 categories according to their involvement in: biological processes, (B) molecular functions, (C) and protein classes. GO terms were used to describe the attributes of the DmHsp22 potential partners.</p

Abundancy of ATP synthase (Complex V) subunits and subunit composition of DmHsp22-associated partners.

<p>Detected subunits of the ATP synthase machinery as mitochondrial partners of DmHsp22 have been summarized in the bar chart from the highest to the lowest percentage of detected proteins. Subunits related to the F1 and F0 sub-complexes have been identified in white and black background bar charts according to their association to the F1 or F0 sub-complexes respectively.</p

Heat shock and recovery does not influence expression levels of DmHsp22.

<p>Confirmation of DmHsp22’s interaction with Hsp60 and Hsp70. (A) HeLa cells expressing DmHsp22 and pcDNA vector were harvested and analyzed for DmHsp22 expression at 0, 6 and 12 hours post-recovery after heat shock by western blot. HS (1 hour heat shock without any recovery), HS+6H recovery (1 hour heat shock at 42°C and 6 hours recovery at 37°C), HS +12H recovery (1 hour heat shock at 42°C and 12 hours recovery at 37°C). anti-Hsp60 was used as loading control. (B) IAC on S1300 g of HeLa cells expressing DmHsp22 as well as HeLa cells expressing control vector under heat shock (HS) conditions, where cells were treated at 42°C for 1 hour followed by 6 hours post-recovery at 37°C and detecting Hsp70 and Hsp60. Results are representative of 4 separate experiments (N = 4).</p

DmHsp22 is an important factor in thermotolerance under heat stress condition.

<p><b>(A)</b> The DmHsp22 (black) and control vector (gray) transfected cells in cells transiently expressing mitochondrial luciferase were cultured and transfected as described in Materials and Methods. Cells were exposed to 40, 42, 44 or 46°C for 30 and (B) 60 minutes in presence of 20 μg. mL^-1 cycloheximide. Following heat exposure, cells recovered for 6 hours at 37°C, were lysed and assayed for luciferase activity. Data are presented as means and SD of four replicates. P*< 0.05; P** < 0.01 and P***<0.001 according to two-way ANOVA (A-B). (C) Fluorescence microscopy of HeLa cells expressing recombinant luciferase. Top 2 rows represent Non-HS cells and bottom 2 rows represent HS cells at 44°C for 30 and 60 min.</p

Proteins interacting with DmHsp22 by orbitrap fusion LC-MS/MS mass spectrometer.

<p>Proteins interacting with DmHsp22 by orbitrap fusion LC-MS/MS mass spectrometer.</p

Characterization of DmHsp22WT and DmHsp22WT-Flag expressing cells.

<p>(A) HeLa cells were transfected with 3 μg of DmHsp22WT, WT-Flag and pcDNA as negative control (Ctrl) and 3 μl of lipofectamine as transfection reagent at 1:1 ratio for 24 hours. Cells were lysed with RIPA buffer and western blot was performed. DmHsp22’s expression level was detected in immunoblot probed with anti-DmHsp22 antibody. Anti-Hsp60 was used as loading control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193771#sec002" target="_blank">Materials and Methods</a>). (B) Percentage of transfected cells (% of total cells) was determined 24 hours after transfection using immunofluorescence. Results are shown as a bar graph (P>0.05). Values represent the means ± SD from ten independent experiments. (C) Whole-cell extracts (WCE), cytosolic extracts (S1300 or S12000g), pellets (P1300g) and purified mitochondria fractions (P12000 or Mito) from HeLa cells transfected with DmHsp22WT and WT-Flag. 25 μg of each fraction were analysed by SDS-Page followed by immunoblotting with Anti-Hsp60 and Anti-cytochrome c as mitochondrial markers and anti-Hsp90 as a cytosolic marker. (D) HeLa cells expressing DmHsp22WT and C-terminal Flag-tagged DmHsp22 were processed for immunofluorescence detection of DmHsp22 as described in Materials and Methods. 24 hours post-transfection, fixed and permeabilized cells were stained with anti-Hsp22 antibody followed by Cy2 (green fluorescence) and DAPI (blue fluorescence) for nuclei labelling. Images were obtained using a fluorescence microscope. (E) Expression of DmHsp22WT and WT-Flag did not change levels of Hsp60 and Hsp70 in immunoblots probed with the corresponding antibodies. Anti-Hsp90 was used as loading control.</p

Forward and reverse primers applied to the constructs of pcDNAHsp22 wild type, pcDNAHsp22WT-Flag and pcDNA-Flag.

<p>Forward and reverse primers applied to the constructs of pcDNAHsp22 wild type, pcDNAHsp22WT-Flag and pcDNA-Flag.</p

Mitochondrial oxygen consumption in intact cells and cellular ATP concentration.

<p>(A) ROUTINE, LEAK and ETS respiration rates measured in HeLa cells transfected with DmHsp22 (blue) as compared to the cells transfected with empty vector (red). Only ETS respiration was significantly higher in transfected cells (N = 7). (B) The respiratory control ratio (RCR = ETS/LEAK) is not different between HeLa cells transfected with DmHsp22 compared to the control cells (N = 7). (C) Cellular ATP concentration due to mitochondrial phosphorylation of ADP into ATP is higher in cells transfected with DmHsp22 as compared to the control cells (N = 4). Data are presented as means ± SEM. P*< 0.05; P** < 0.01, according to t-test.</p