15 research outputs found
Elucidating the role of free polycations in gene knockdown by siRNA polyplexes
Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8. kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8. kDa (bPEI 1.8. kDa) and 25. kDa (bPEI 25. kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8. kDa and bPEI 25. kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8. kDa improves the siRNA delivery properties, but with markedly different formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25. kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25. kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH. Statement of Significance: Gene silencing therapy has the potential to treat diseases which are beyond the reach of current small molecule-based medicines. However, delivery of the small interfering RNAs (siRNAs) remains a bottleneck to clinical implementation, and the development of safe and efficient delivery systems would be one of the most important achievements in medicine today.A major reason for the lack of progress is insufficient understanding of cell-polyplex interaction. We investigate siRNA delivery using polyethyleneimine (PEI) based vectors and examine how crucial formulation parameters determine the challenges associated with PEI as a delivery vector. We further evaluate how lipid conjugation of PEI influences formulation, cytotoxicity and polymer interaction with cells and cargo as well as the proton sponge capabilities of the vectors
CD and MPR are reduced in α-syn expressing SH-SY5Y cells.
<p>(A, B, C) Confocal microscopy analysis of single immunostaining for CD, MPR300 and Lamp1, respectively, in Ctrl and α-syn expressing SH-SY5Y cells (ASO). Scale bar 8 μm. Panels are representative of 3 different experiments. Images were also acquired on an automated Olympus microscope and the number and the area (D) of the vesicles pr. nuclei quantified using Scan^R imaging software (Olympus) (three slides per condition). (N = 5; Two tailed T-test) *P<0.05 vs Ctrl. The total number of vesicles was normalized to DAPI positive cells and expressed as media of vesicles per cell. (E) WB analysis for proCD and CD cleaved peptides migrating at 32kDa and 14kDa, MPR300, Lamp1 and α-syn from total lysate (30μg) of human stable-transfected α-syn overexpressing cells (ASO) and their correspondent empty-vector control (Ctrl) cells. A representative picture showing the levels of α-syn in each sample is shown below. The optical density analysis is reported below. Data were normalized on the basis of the correspondent β-actin values and expressed as % of Ctrl. The experiments were run 3 times in triplicate. (N = 3; Two tailed-T Test). (*) p<0.05 and (**) p<0.01 vs Ctrl. (F) Ctrl and stable-transfected α-syn SH-SY5Y cells were treated with triple-labelled pH-sensitive nanosensors for 24 h and imaged by confocal microscopy. The ratio of the intensity signals from the pH-sensitive and reference fluorophores was converted to pH via the calibration curve. To ensure the assay was working, an increase in vesicular pH was induced in Ctrl and ASO cells by treatment with 100 nM Bafilomycin A<sub>1</sub> that blocks the vacuolar-type H<sup>+</sup>-ATPase. Mean ± SEM (number of slides n = 10) are presented. A maximum of 7% and 0.5% of measurements fell outside the range of the nanosensor for the bafilomycin A<sub>1</sub>-treated and untreated samples, respectively. Each experiment was repeated three times with similar results and for each experiment between 800 and 1000 data points were acquired for every cell line.</p
AP1 protein levels are reduced in α-syn overexpressing mice cortex.
<p>(A) Western blot analysis of AP-1, AP-2, AP-3, SorLA, Sortilin and VPS35 from WT and ASO<sup><i>Tg/Tg</i></sup> cerebral cortical tissues. Optical density analysis is reported below. Data were normalized on the basis of the correspondent β–actin values and expressed as % of WT. The experiments have been run 5 times in triplicate (N = 5; Two tailed-T test) (**) p<0.01 vs. WT. (B) Confocal microscopy analysis of Lamp-2 surface staining on Ctrl and ASO cells with anti-Lamp2 antibody. Cells are stained with the primary antibody before permeabilization.</p
MPR300 is decreased in human brain from patients with early PD.
<p>WB of MPR300 and α-syn in SDS-soluble protein lysates generated from the anterior cingulate cortex of 6 autopsy-confirmed patients with Braak Lewy body stage 4 (PD) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160501#pone.0160501.ref065" target="_blank">65</a>] versus 9 controls as detailed in the table below reporting their demographic details. Immunoblot data was normalized to β-actin and a multivariate analysis performed to determine if MPR300 or α-syn were changed in PD. Age and post-mortem delay were included as covariates. The results show a significant reduction in MPR300 protein and an expected significant increase in α-syn levels in PD anterior cingulate cortex. Values are given as mean ± SEM and * = p<0.05.</p
CD and MPR300 protein levels are reduced in α-syn overexpressing mice cortex.
<p>(A) WB analysis for α-syn performed in total lysate from cerebral cortical tissues of 3, 6 and 12 month-aged WT, ASO<sup><i>Tg/+</i></sup> and ASO<sup><i>TgTtg</i></sup> mice. WT tissues are from C57BL/6J mice (background matched to ASO<sup><i>Tg/Tg</i></sup><i>)</i>. No obvious differences in α-syn expression were detected between the two WT mice with different background. N = 5; One-way Anova with post-hoc Tukey test. (B, C) WB for MPR300 and proCD/CD in total lysate from cortex of heterozygous α-syn ASO<sup><i>Tg/+</i></sup> and homozygous α-syn ASO<sup><i>Tg/Tg</i></sup> mice respectively and their correspondent background matched controls. Charts of the band optical density (OD) are shown below each panel. Data were normalized to the corresponding β-actin values and expressed as % of WT. (*) p<0.05 vs WT. The experiments were run 5 times in triplicate. N = 5. Two tailed T-Test.</p
MPR300 preferentially localizes in late endosomes in ASOTg/Tg neurons.
<p>(A-C) Confocal microscopy analysis of MPR300 (green) and EEA1, TGN46 and Rab7 (red), respectively, in WT and ASO<sup><i>Tg/Tg</i></sup> cortical neurons. Scale bar 5 μm. (D) The extent of MPR300 co-localization to EEA1, TGN46 and Rab7 is reported in panel D. Quantitative analysis was performed using Zen software. The (R) coefficient (Pearson’s coefficient) was used for the quantitative and comparative analyses (R). Data are expressed as mean ± SEM. n = 8. *p<0.05. Two tailed T-Test).</p
Schematic drawing of MPR300 trafficking in cells.
<p>The top panel displays how MPR300 traffics in neurons under normal physiological conditions. In short: after final glycosylation in the Golgi, MPR300 can either be secreted by a default route to the cell membrane (1) or actively transported to the endo-lysosomal system by interacting with AP-1 and GGA 1–3 (2). MPR300 on the membrane can be internalised via a coated pit to reach the endosomes (3). When MPR300 reaches endosomes, the retromer complex and AP-1 facilitates the retrograde transport to the TGN (4). More than 90% of endogenously expressed MPR300 is estimated to enter the TGN-endosome shuttle at any given time and this transport is essential for the transport of CD to lysosomes (5). α-Syn enters the lysosomes via autophagosomes (6). The lower panel reports how a reduced MPR300 retrograde transport in neurons likely influences proCD sorting in PD neurons (4). The reduction of MPR300 reduces the transport of proCD to the endosomal system and its processing into the mature form of CD. Moreover, MPR300 accumulates in the membrane of late endosomes and in the intraluminal vesicles (ILV). Finally, the only escape from endosomes is by lysosomal degradation or secretion (in this latter case the ILV are termed exosomes).</p
The reduction in MPR300 levels is partially controlled by the exposure to lysosome inhibitors.
<p>(A) Confocal microscopy analysis of MPR300 (green) and Lamp1 (red) positive vesicles. To counteract the decrease of MPR300 vesicles, Ctrl and ASO cells were exposed to vehicle, Chloroquine (10mM) and NH<sub>4</sub>Cl (5mM) for 6 hrs. Images were also acquired on an automated Olympus microscope and the number of the vesicles quantified using Scan^R imaging software (Olympus) (three slides per condition) (B). (N = 3; One-way Anova with post-hoc Tukey test) *P<0.05 vs Ctrl. The extent of MPR300 co-localization to Lamp1 positive vesicles is reported in panel C. Quantitative analysis was performed using Zen software. The (R) coefficient (Pearson’s coefficient) was used for the quantitative and comparative analyses (R). Data are expressed as mean ± SEM. n = 4. *p<0.05 One-way Anova with post-hoc Tukey test).</p
MPR300 protein levels are significantly increased in the soluble fraction of ASO<sup>Tg/Tg</sup> cortical tissues and in ASO cells.
<p><b>(A) Media collected from ASO cells and the corresponding control (Ctrl) samples and from ASO and WT cortical neurons (after 10 days in culture) were ultra-centrifuged at 100,000 rpm for 12 h. The pellets were solubilized in 2% SDS-loading buffer and (1/10 of the total amount) analysed by WB for MPR300 and proCD. Total lysate was loaded and analysed on the same membranes. proCD was not detectable under our experimental conditions (data not shown). The WB is representative of 3 different experiments. Densitometric analysis of MPR300 levels in media from ASO cells and neurons is reported below. Data were normalized on the basis of Hsc70 levels. The experiments were run 3 times in triplicate (N = 3; Two-tailed-T test) (***) p<0.005 vs. WT.</b> (B) WB analysis for MPR300 and proCD were performed in the soluble and insoluble fractions from cortical tissues of ASO<sup><i>Tg/Tg</i></sup> mice and the correspondent WT samples. Hsc70 (soluble and/or secreted marker protein) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160501#pone.0160501.ref063" target="_blank">63</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160501#pone.0160501.ref064" target="_blank">64</a>] (protocol is described in Methods) and Lamp1 blot were used as respective positive and negative controls of the soluble and insoluble fractionations. The lack in Lamp1 blot signals in the media of cells and neuronal cultures indicates that there was no cell leakage under our experimental conditions. Optical density analysis is reported below. Data were normalized on the basis of Hsc70 (soluble) or Lamp1 (insoluble) levels and expressed as % of WT. The experiments were performed 4 times in triplicate (N = 4; Two tailed-T test) (*) p<0.05 and (**) p<0.01 vs. WT.</p