A role for intracellular PrP^C retention in neuronal dysfunction.

<p>(A) PrP^<b>C</b> on the plasma membrane (PM) influences the activity of neurotransmitter receptors, ion channels, and signaling complexes with which it interacts. (B) Owing to retention in transport organelles (ER/Golgi), misfolded/aggregated PrP^<b>C</b> sequesters the interacting protein in intracellular compartments, leading to loss of normal function on the cell membrane [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004745#ppat.1004745.ref006" target="_blank">6</a>]. Intracellular retention might also cause the complex to function abnormally and generate a toxic signal.</p

A Role for PrP^C Function in Prion Diseases

<p>PrP^C on the cell surface performs its normal function by associating with a hypothetical transmembrane interactor (“X”). In the disease state, PrP^Sc (or a misfolded intermediate) initiates a toxic signal by associating with different interactors (“Y” or “Z”), possibly as a result of oligomerization.</p

Two Roles for PrP in Cell Adhesion

<p>(A) In wild-type zebrafish (left), PrP-1 promotes proper delivery of E-cadherin from the Golgi to the plasma membrane (PM), possibly via activation of a signal transduction cascade involving Src-family tyrosine kinases. In morphant fish lacking PrP-1 (right), E-cadherin accumulates in intracellular vesicles, resulting in reduced delivery to the plasma membrane. As a result, Ca⁺²-dependent, cadherin-mediated cell adhesion is impaired. (B) PrP molecules on adjacent cells undergo homophilic interactions that promote cell adhesion in a Ca⁺²-independent manner, at the same time generating an intracellular signal involving tyrosine phosphorylation. The PrP functions depicted in the two panels of this figure could be linked, if the intracellular signal generated by homophilic binding of PrP molecules (B) regulates cadherin trafficking (A).</p

SS14 <i>gemini</i> surfactant molecule and evaluation of transfection effectiveness of SS14-containg liposome formulations.

<p>(A) Chemical structure and space-filling molecular model of <i>gemini</i> surfactant SS14. Color coding: yellow  =  sulfur; purple  =  nitrogen; grey  =  carbon; white  =  hydrogen. (B) Cytotoxicity (viability, left axis, white bars) and transfection efficiency (% of EGFP-positive cells, right axis, grey bars) of DOPC/DOPE/SS14 (25∶50∶25 molar ratio) lipoplexes on U87-MG cell line as a function of charge ratio (CR, +/−). (C) Cytotoxicity and transfection efficiency of binary DMPC/SS14, DOPC/SS14 (75∶25 molar ratio each), ternary DMPC/DMPE/SS14, and DOPC/DOPE/SS14 (25∶50∶25 molar ratio each) lipoplexes at CR5 on U87-MG cell line. Lipofectamine 2000 was used as positive control in transfection experiments. All results are expressed as mean ± SEM (n = 3).</p

Complexation abilities of DOPC/DOPE/SS14 liposome formulations and evaluation of their transfection effectiveness.

<p>(A) Fluorophore-exclusion titration of DOPC/DOPE/SS14 liposomes at 29.2∶58.3∶12.5 (grey circles), 25∶50∶25 (black rhombus), and 16.7∶33.3∶50 molar ratios (red triangles) as a function of CR. All curves underlying data simply represent a guide to the eye and were drawn to better evidence trend variations. Cytotoxicity (viability, left axis, white bars) and transfection efficiency (% of EGFP-positive cells, right axis, grey bars) of the three different DOPC/DOPE/SS14 lipoplexes at CR5 on COS-7 (B), U87-MG (C), and MG63 (D) cell lines at CR5. Results are expressed as mean ± SEM (n = 3). Examples of cytofluorimetric analysis are reported as FL1 (green fluorescence) <i>vs.</i> FL2 (orange fluorescence) dot plot of U87-MG transfected cells (C, upper panels). Mock-transfected (pCMV-GLuc) but autofluorescent population of cells lies along the 0, 0; 10⁴, 10⁴ diagonal. EGFP-expressing cells appear as an additional population delineated by region 2 (R2), where FL1>FL2.</p

Hydrodynamic diameter, <i>ζ</i>-potential and polydispersity index (P.I.) of each liposome formulation.

a<p>Mean ± Standard Deviation.</p

GSH-mediated lipoplex disassembly in batch and effect of intracellular GSH levels on transfection efficiency.

<p>(A) Stability of DOPC/DOPE/SS14 (16.7∶33.3∶50 molar ratio) lipoplexes at CR5 in presence of GSH or GSSG. Results are presented as % of fluorescence emitted with respect to DNA. (B) Experimental procedure. MG63 cells were divided in four groups: untreated CTRL, BSO-, NAC-, and Vit-C-treated cells. Following pharmacological treatment (t₀), cells underwent 48 h transfection (t_final) with DOPC/DOPE/SS14 (16.7∶33.3∶50 molar ratio) lipoplexes at CR5. Oxidative stress and GSH content were measured at t₀ ((C) and (E), respectively) and after transfection ((D) and (F), respectively). Transfection efficiency, expressed as % of EGFP-positive cells, was also evaluated (G). A linear correlation between GSH content and transfection efficiency was observed (H). Results are expressed as mean ± SEM (n = 3). $ <i>p</i><0.05 <i>vs.</i> CTRL; * <i>p</i><0.05 <i>vs.</i> BSO; § <i>p</i><0.05 <i>vs.</i> NAC; £ <i>p</i><0.05 <i>vs.</i> Vit-C.</p

Hydrodynamic diameter, <i>ζ</i>-potential and polydispersity index (P.I.) of DOPC/DOPE/SS14 liposomes and lipoplexes at CR5.

a<p>Mean ± Standard Deviation.</p

Untranslocated PrP shows cytosolic localization.

<p>Hippocampal neurons from C57BL/6J mice were transfected with a plasmid encoding PrP-EGFP (green). Twelve days after transfection cells were exposed to the vehicle (A) or treated with 10 μM CAM741 for 24 h plus 5 μM MG132 during the last 6 h (B–D). Cells were then fixed and reacted with DAPI (blue) to stain the nuclei. Cells in C and D were also immunostained with an anti-PDI or anti-golgin antibody (red) to visualize the ER and Golgi, respectively. Scale bar  = 10 μm in A (also applicable to B), and 5 μm in C (also applicable to D).</p

Proteasome inhibitors do not induce ER stress in primary neurons.

<p>Cortical (A) or cerebellar granule neurons (B) were treated with 5 μM MG132 or epoxomicin (Epoxo) for the times indicated, with 5 μM MG132 and 10 μM CAM741 for 18 h, or 5 μg/ml tunicamycin (Tm) for 8 h. After treatment, total RNA was extracted and analyzed by reverse transcription-PCR. XBP1 splicing was determined by the appearance of rapidly migrating spliced XBP1 in tunicamycin-treated cells. The arrows point to unspliced (uXBP1) and spliced (sXBP1) transcripts.</p