Single section Golgi analysis reveals alterations in dendritic branching and spine formation in the PFC following 7 days of abstinence from cocaine.

<p>(<b>A</b>) Representative photomicrograph of a Golgi stain pyramidal neuron and tracing from yoked-saline control (top left, top right) and cocaine (bottom left, bottom right) treated rats scale bar  = 50 µm (<b>B</b>) Sholl plot analysis of animals self-administering saline (open circles) and cocaine (black-filled circles) (<b>C</b>) Representative basal dendritic segment from a yoked-saline (top) and cocaine (bottom) neuron scale bar  = 10 µm (<b>D</b>) Quantitative analysis of total spine number and spine density classified by spine sub-type from 4–5 segments from 5–7 neurons from each animal, n = 4 animals/group. *, <i>p</i><0.05; ***, <i>p</i><0.001.</p

Spine loss in rTg4510 mice is independent of calcium dysregulation.

<p>High-resolution in vivo images of dendrites and spines from control (A) and rTg4510 (B) show substantial loss of spines in rTg4510 mice. For better visualization, the image background was removed and the contrast adjusted. Analysis of dendritic spine densities was performed on raw images. The average dendritic spine density (C) was significantly decreased in rTg4510 mice when compared to controls (*, p < 0.05). Spine density in rTg4510 mice did not significantly correlate with YC ratios in the parental dendrite (D). Scale bars (A, B) represent 5 μm. </p

Array tomography reveals alterations in synapse density in the PFC following 7 days of abstinence from cocaine.

<p>(<b>A</b>) Ribbons of 70 nm sections were stained with postsynaptic marker PSD95 (red), presynaptic marker synaptophysin (green), and DAPI to label nuclei (blue) (<b>B</b>) Another set of ribbons were stained with synaptophysin (green) and GAD65 (magenta) to determine the percentage of inhibitory synapses. Raw images of single sections from saline (left) and cocaine treated (right) rats are shown in the top row of panels <b>A</b> and <b>B</b>. Images were aligned, processed to remove noise, and synapses detected in three-dimensional volumes as shown in the 3D rendering of 5 serial sections (bottom rows <b>A</b> and <b>B</b>) (<b>C</b>) Quantification of presynaptic terminals (<b>D</b>) Quantification of PSD95 positive postsynaptic terminals (<b>E</b>) Quantification of GAD65 positive terminals. Graphs show mean + S.E.M. * p<0.05 t-test; n = 5 animals/group; scale bar  = 10 µm top panels, and 2 µm bottom panels.</p

Yellow cameleon imaging in rTg4510 and control mice.

<p>In vivo multiphoton imaging of somatosensory cortex neurons expressing the ratiometric calcium indicator YC3.6 (A) demonstrates that the indicator fills both dendrites (B, blue arrow heads) and dendritic spines (B, yellow arrow heads). Calcium concentration images from control (C) and rTg4510 (D) mice were calculated from YC ratio (YFP/CFP) images and color-coded according to the color gradient shown in the middle. Scale bars (B, C, D) represent 10 μm.</p

Calcium concentrations in dendrites and spines are not disrupted by tau over-expression.

<p>Average YC ratios (YFP/CFP) and calcium concentrations recorded from dendrites and dendritic spines in control and rTg4510 mice (A). Distributions of YC ratios in dendrites and dendritic spines from control (B, C) and rTg4510 mice (E, F) show no significant difference between control and rTg4510 mice. The dashed vertical lines indicate calcium overload thresholds at the 95^th percentile of the control mice data, determined for dendrites and spines separately. Data are shown as mean ± standard deviation. </p

FLIM-FRET study of ApoE conformation and Aβ-ApoE interaction reveals multiple aspects of ApoE4 associated plaque pathology.

<p>Inter-epitope distances are normalized to the Förster radius. a) A dense core senile plaque from the cortex of a patient homozygous for ApoEε3. Aβ (green) and ApoE NT (red) are extremely well co-localized which illustrates that the plaque is decorated with ApoE. b) Schematic showing the three FLIM-FRET measurements that were made. We independently measured the interacting fraction and distance between Aβ and both ApoE terminal domains as well between the two ApoE domains. c) ApoE CT is in closer apposition to Aβ than ApoE NT, consistent with the assumption that the hydrophobic lipid binding region interacts with Aβ. The difference in distance is small enough to suggest that ApoE envelops Aβ in a similar fashion than it is known to interact with lipids. d) A significantly greater proportion of Aβ is bound to ApoE in the case of ApoE4. The data suggest a reduced capacity of ApoE4 to induce clearance of Aβ. e) ApoE4 has a slightly tighter terminal interaction. This is surprising because a large difference in inter-terminal interaction is expected from the <i>in vitro</i> data. f) ApoE shows a significantly lower numbers of interacting terminal domains. These data are proof that ApoE4 undergoes a greater amount of cleavage either before or after binding to Aβ. Differential cleavage may mediate Aβ clearance or deposition.</p

Western blots of brain homogenates from AD patients and normal aged brains.

<p>a) Using poly-clonal antibody, the distribution of ApoE fragments is clearly different between healthy aged brains and AD brains with further marked differences between genotypes. b) Comparisons of densitometry show significant increases in the amount of ApoE in Alzheimer brains, with the greatest amount in individuals homozygous for ApoE4. c) We also measured an increase in LMW (7–10 kDa) fragments in the case of AD, the presence of APOEε4 further amplifies the effect. The effect is similar but less subtle for HMW (17–34 kDa) fragments. In all cases asterisks indicate significance as assessed using ANOVA and Bonferonni-Dunn post-hoc test.</p

List of cases used for the FLIM-FRET study.

<p>The genotype, age of the patient at death, sex and postmortem interval (PMI) is given where available from ADRC records. The check marks show which of the three experiments in which the brain was used. Some brains were used in more than one comparison depending on availability of tissue. For each of the 6 comparisons, the total number of plaques imaged (‘n’) is also given.</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

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