5 research outputs found
PGRMC1 mediates the binding of Abeta oligomers to neurons <i>in vitro</i>.
<p>Co-immunolabeling for Abeta oligomer binding (<b>A–C</b>) and sigma-2/PGRMC1 expression (<b>D–F</b>) in the same field of view in hippocampal and cortical cultures (21DIV). Untreated neurons (<b>A</b>, <b>D</b>) exhibit Abeta oligomer binding to synaptic sites on neurites and low levels of sigma-2/PGRMC1 expression. In the presence of siRNA to sigma-2/PGRMC1, both Abeta oligomer binding and sigma-2/PGRMC1 expression are significantly reduced (<b>B, E</b>). Non-targeting siRNA (<b>C, F</b>) has no effect. <b>G. H</b>. Graphs of immunocytochemically detectable PGRMC1 protein expression associated with neuron cell bodies (G) and synaptic puncta (H), and Abeta oligomer binding to synapses for each of nine separate experiments (expressed as a percentage of untreated control culture values mean ± S.E.M.). siRNA-mediated reduction in PGRMC1 protein expression of up to 28% results in a corresponding decrease in Abeta oligomer binding by up to 91% (linear regression for PGRMC1 expression in neuronal cell bodies, r<sup>2</sup> = 0.799, p = 0.0011; for PGRMC1 expression in synaptic puncta, r<sup>2</sup> = 0.554, p = 0.02).</p
C-terminal antibodies directed against the C-terminus of PGRMC1 prevent (A–D) and displace (E–H) Abeta oligomer binding to neurons and glia.
<p>Abeta oligomers bind to a subset of neurons and glia in mature hippocampal primary neurons 21DIV (<b>A, E, red bar in I</b>) compared to vehicle-treated (no Abeta) cultures (<b>B, F</b>, blue bar in <b>I</b>). Graphs in <b>I</b> are average of 3 experiments (avg. intensity of Abeta oligomer puncta + S.E.M., expressed as a percentage of Abeta oligomer-treated condition, difference in binding intensity vs. Abeta oligomer condition *p<0.05, Student's t-test). Abeta oligomer binding to cultured neurons is significantly reduced in the presence of C-terminal antibody to sigma-2/PGRMC1 regardless of whether it is added before (<b>D</b>, green bar in <b>I</b> [prevention], 58% reduction) or after (<b>H</b>, green hatched bar in I [treatment], 26% reduction) oligomers. This suggests that oligomers are competitively displaced from receptors at synaptic sites. Non-immune IgG (<b>C, G</b> and maroon bars in <b>I</b>) and an N-terminal antibody to sigma-2/PGRMC1 (data not shown) cannot reduce oligomer binding under either condition. <b>J</b> Effects of antibodies on membrane trafficking rate in the presence or absence of Abeta oligomers (expressed as a percentage of vehicle-treated in the absence of Abeta, difference in trafficking rate vs. Abeta oligomer- or vehicle-treated condition *p<0.05, Student's t-test). The C-terminal antibody directed against amino acids 185–195 in sigma-2/PGRMC1 does not rescue oligomer-induced deficits, but induces trafficking deficits on its own in the absence of Abeta oligomers, pointing to a critical role of this protein in normal membrane trafficking.</p
Anti-Abeta compounds are ligands for sigma-2/PGRMC1 receptor.
<p><b>A</b>, CT0109, CT0093, CT01344 and CT01346 displace the fiduciary sigma-2 ligand [<sup>3</sup>H]-DTG from receptors on human B cell lines. <b>B</b>. Autoradiograms of 18.4 nM [<sup>125</sup>I]RHM-1 binding to human frontal cortex slices in the presence of 10, 100, 1000, 10,000 nM of CT0109 and CT0093, N = 4. Color bar under images show false coloring scale. [<sup>125</sup>I]RHM-1 displays specific saturable binding to human frontal cortex tissue as assessed by quantitative autoradiography in dose-response format (<b>C</b>) and as a Scatchard plot (<b>D</b>). <b>E</b>. Dose response curves for data obtained from autoradiograms in <b>B</b>. The Ki's for CT0109 and CT0093 at the [<sup>125</sup>I]RHM-1 binding site were 57±23 nM and 33±12 nM, respectively.</p
CogRx sigma-2/PGRMC1-selective small molecules are functional antagonists.
<p><b>A, B</b> Sigma-2/PGRMC1 agonist siramesine causes dose-dependent activation of caspase 3 in primary neuronal cultures (<b>A</b>) and in SKOV-3 human ovarian cancer cells (<b>B</b>) but sigma-2/PGRMC1 antagonists RHM-1, CT0109 and CT0093 do not. <b>C</b>, <b>D</b> Sigma-2/PGRMC1 agonists siramesine, WC-26 and SV-119 cause dose-dependent cell death in primary hippocampal/cortical cultures (<b>C</b>) and in SKOV-3 human ovarian cancer cells (<b>D</b>) but sigma-2/PGRMC1 antagonists RHM-1, CT0109 and CT0093 do not, except at very high concentrations (>100 µM). (<b>E</b>) Treatment of cultures of hippocampal and cortical cells with 20 to 80 µM SV-119 for 24 hours induced the activation of caspase 3/7 (*p<0.05 by 2-tailed Student's t-test compared to control). Co-treatment of cultures with 40 µM CT0109 or CT0093 did not increase caspase activity and blocked the activation by the agonist SV-119.</p
Sigma-2/PGRMC1 protein localizes to synaptic puncta on mature primary hippocampal cultures (21 days <i>in vitro</i>) and expression levels are positively correlated with Abeta oligomer binding.
<p>sigma-2/PGRMC1 (<b>A–D</b>, red) is expressed at low levels in untreated cultures and is localized in cell bodies of neurons and glia, in neurite shafts, and adjacent to presynaptic puncta (<b>A–D</b>, synaptophysin  =  green) <b>B</b>. 66.7%±2.4 (average ± S.E.M., N = 110 neurons) of PGRMC1 positive puncta on neurons co-localize (yellow) with synaptophysin positive puncta. <b>E–L</b>. Positive correlation between sigma-2/PGRMC1 expression and Abeta oligomer binding in neurons (Abeta oligomers  = 400 nM, 1 hour treatment). <b>E–H</b>. Only one neuron (MAP2 positive arrow #1 in <b>E–H</b>) in this field is labeled with punctate Abeta oligomer binding (<b>G</b>), and exhibits elevated PGRMC1 expression (<b>H</b>, 3.3×10<sup>5</sup> RFU) compared to surrounding neurons (#2 = 1.6×10<sup>5</sup>, #3 = 1.8×10<sup>5</sup> RFU). <b>I–L</b>. Vehicle-treated cultures express a similar range of sigma-2/PGRMC1 expression in neurons (arrow #1 in I  = 2.62×10<sup>5</sup>, #2 = 1.21×10<sup>5</sup> RFU). All scale bars  = 20 microns. <b>M</b>, Binning neurons according to their intensity of sigma-2/PGRMC1 immunofluorescence and graphing the average values for Abeta binding from each bin reveals a positive correlation between the intensity of Abeta oligomer binding to synaptic puncta and the expression of sigma-2/PGRMC1 in the cell body that is significant (Kruskal-Wallis, p<0.001). N. A similar analysis of sigma-2/PGRMC1 imunofluorescence in the synaptic puncta also shows a positive correlation with Abeta oligomer binding intensity to synaptic puncta (Kruskal-Wallis p<0.001).</p