Targeting caspase-3 as dual therapeutic benefits by RNAi facilitating brain-targeted nanoparticles in a rat model of Parkinson's disease

Abstract

The activation of caspase-3 is an important hallmark in Parkinson's disease. It could induce neuron death by apoptosis and microglia activation by inflammation. As a result, inhibition the activation of caspase-3 would exert synergistic dual effect in brain in order to prevent the progress of Parkinson's disease. Silencing caspase-3 genes by RNA interference could inhibit the activation of caspase-3. We developed a brain-targeted gene delivery system based on non-viral gene vector, dendrigraft poly-L-lysines. A rabies virus glycoprotein peptide with 29 amino-acid linked to dendrigraft poly-L-lysines could render gene vectors the ability to get across the blood brain barrier by specific receptor mediated transcytosis. The resultant brain-targeted vector was complexed with caspase-3 short hairpin RNA coding plasmid DNA, yielding nanoparticles. In vivo imaging analysis indicated the targeted nanoparticles could accumulate in brain more efficiently than non-targeted ones. A multiple dosing regimen by weekly intravenous administration of the nanoparticles could reduce activated casapse-3 levels, significantly improve locomotor activity and rescue dopaminergic neuronal loss and in Parkinson's disease rats' brain. These results indicated the rabies virus glycoprotein peptide modified brain-targeted nanoparticles were promising gene delivery system for RNA interference to achieve anti-apoptotic and anti-inflammation synergistic therapeutic effects by down-regulation the expression and activation of caspase-3.

Figure 1. In vivo imaging of fluorescent labeled DPR/DNA NPs.

(A) In vivo distribution in mice with different treatment (Left: DGLs/DNA NPs; Middle: DPR/DNA NPs; Right: without treatment) at 15 min after intravenous administration in Balb/c mice. (B) In vivo distribution in mice with different treatment (Left: DGLs/DNA NPs; Middle: DPR/DNA NPs; Right: without treatment) at 1 h after intravenous administration in Balb/c mice. (C) 3D fluorescent localization in DPR/DNA NPs injected mouse skeleton with CT scanning.

Figure 2. The localization of NPs in midbrain and the permeability of ¹²⁵I-labeled NPs across BCECs monolayer.

Brain sections of NP injected rat were immunostained with (A) anti-Factor VIII polyclonal antibody labeled brain capillaries and (D) anti-Neurofilament monoclonal antibody labeled neurons. (B) and (E) were images of NPs distribution. (C) was the merged image of (A) and (B), while (F) was that of (D) and (E). The circles indicated the colocation of NPs and related labeled cells. Red: Alexa Fluor 555 labeled secondary antibody; Green: BODIPY labeled DPR/DNA NPs; Yellow: Merged signal of red and green. Original magnification: ×200. (G) The permeability of ¹²⁵I-labeled NPs across BCECs monolayer. Significance: *, p<0.05; **, p<0.01, significance represents DPR/DNA NPs vs. DP/DNA NPs.

Figure 3. Evaluation of activated caspase-3 in rotenone induced Parkinson’s disease rat model.

(A) Caspase-3 mRNA silencing percentage by RT-PCR in brains at various days of rotenone treatment in rats with different NPs. Data are expressed as mean±S.E.M (n = 3). Significance: *p<0.05; ***p<0.001, significantly different as compared between two groups. (B) Activated caspase-3 comparison by western blot in brains at various days of rotenone treatment in rats with different NPs. (C) Quantitative evaluation of activated caspase-3 positive immunofluorescence at cortical layer, substantia nigra, hippocampus and caudate putamen and in rats with rotenone treatment for 45 days.

Figure 4. Behavioral changes of rotenone treated rats with different NPs injection.

(A) Number of line crossing. (B) Retention time. Data are expressed as mean±S.E.M (n = 10). Significance: **p<0.01; ***p<0.001, significantly different as compared to saline injected rats with rotenone treatment (the negative control group).

Figure 5. TH-immunoreactivity in the substantia nigra.

Representative immuno-staining sections selected from saline (A, D, G) and different NP injected groups (DPR/pshSc NPs: B, E H; DPR/pshC-3 NPs: C, F, I) with rotenone treatment for (A, B, C) 10 days, (D, E, F) 25 days and (G, H, I) 45 days. (J) Stereological counting of TH positive nigral dopamine neurons following saline and different NP injected groups (DPR/pshSc NPs and DPR/pshC-3 NPs) with rotenone treatment for 10 days, 25 days and 45 days. * p<0.05; ** p<0.01.

Figure 6. TUNEL staining on frozen sections of brain.

(A–I) substantia nigra; (J–R) caudate putamen. Representative sections selected from saline and different NP injected groups with rotenone treatment for 10 days, 25 days and 45 days.

Figure 7. The effects of rotenone treatment on inflammatory related markers.

(A) pro-inflammatory cytokine TNF-α level and (B) NO level were examined in midbrain tissues (mainly substantia nigra) with various length of rotenone treatment injected with saline or different NPs. Data are expressed as mean±S.E.M (n = 3). Significance: *p<0.05; **p<0.01, significantly different as compared between two groups.