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. 2018 Feb 7;26(2):550-567.
doi: 10.1016/j.ymthe.2017.11.015. Epub 2017 Nov 29.

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson's Disease

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Free PMC article

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson's Disease

Diana Alarcón-Arís et al. Mol Ther. .
Free PMC article

Abstract

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.

Keywords: 5-HT neurotransmission; ASO; DA neurotransmission; Parkinson’s disease; caudate putamen; intranasal administration; prefrontal cortex; siRNA; α-synuclein.

Figures

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Figure 1
Figure 1
499-siRNA and 1233-ASO Molecules Selectively Reduce α-Synuclein Expression in Cultured Cells (A and G) Comparative alignment showing the oligonucleotide sequence of human Snca, Sncb, and Sncg. Green letters indicate nucleotide differences compared with Snca sequence. Red letters indicate the specific sequence targeted by (A) 499-siRNA or (G) 1233-ASO. (B, C, and E) qRT-PCR quantification of Snca (B), Sncb (C), and Sncg (E) mRNA expression in M17-EV 24 hr after transfection with 200 nM 499-siRNA, MAYO2-siRNA, or SNCA2-siRNA. Cells transfected with nonsense siRNA (NS) were used as control. Target gene expression was normalized to two different housekeeping genes: GAPDHD and RPLPO. (D) Top: immunofluorescence images showing α-synuclein expression in M17-EV and M17-Syn. Blue: Hoechst staining; green: α-synuclein. Scale bar: 20 μm. Bottom: α-synuclein immunoblot image and quantification confirming the overexpression of α-synuclein protein levels in M17-Syn (n = 3). *p < 0.05, compared with M17-EV cells (one-way ANOVA followed by Tukey’s post hoc test). (F) qRT-PCR quantification of Snca expression in M17-Syn 24 hr after transfection with 499-siRNA, MAYO2-siRNA, and SNCA2-siRNA. Cells transfected with NS-siRNA were used as a control. (H and I) qRT-PCR quantification of α-synuclein expression in (H) M17-EV or (I) M17-Syn cells transfected with 300 nM nonsense 1227-ASO or 1233-ASO for 24 hr. In all graphs, histograms represent average ± SEM. ˆp < 0.05, ˆˆˆp < 0.001, compared with cells treated with lipofectamine alone; +p < 0.05, ++p < 0.01, +++p < 0.001, compared with cells treated with NS-siRNA (one-way ANOVA followed by Tukey’s post hoc test). 499-siRNA, MAYO2-siRNA, or SNCA2-siRNA are siRNA sequences designed to target regions of the Snca mRNA encoding α-synuclein protein (Table S1 shows siRNA sequences). M17-EV, M17 human neuroblastoma cells expressing empty vector; M17-Syn, M17 human neuroblastoma cells overexpressing α-synuclein.
Figure 2
Figure 2
Preferential Accumulation of Indatraline-Conjugated ASO Molecules in Monoamine Neurons after Intranasal Administration (A) Mice were intranasally administered with Alexa 488-labeled indatraline-conjugated 1233-ASO targeting α-synuclein (A488-IND-1233-ASO) or Alexa 488-labeled indatraline-conjugated nonsense 1227-ASO (A488-IND-1227-ASO) at 30 μg/day for 4 days and sacrificed 6 hr after last administration (n = 3 mice/group). Confocal images showing the co-localization of A488-IND-ASOs (yellow) with TH+ neurons (red) in the SNc/VTA and LC or with TPH2+ neurons (red) in the DR identified with white arrowheads. Cell nuclei were stained with DAPI (blue). Scale bars: 10 μm. (B and C) Bars show the extracellular concentration of indatraline-conjugated 1233-ASO targeting α-synuclein (IND-1233-ASO) (B) and 1233-ASO molecules (C) (expressed as 109 molecules/μL) in DR, LC, SNc/VTA, and CPu. Mice received two consecutive ASO doses (1000 μM to 60 μg and 3000 μM to 180 μg) intranasally administered with time intervals of 1 hr. Note the higher extracellular IND-1233-ASO concentration in the DR compared with all brain areas. Data are mean ± SEM. ***p < 0.001 versus brain areas (two-way ANOVA followed by Tukey’s post hoc test). (D) Proposed mechanisms for transport of indatraline-conjugated oligonucleotides to brain following intranasal administration. Three potential pathways have been indicated for compound transport to the olfactory bulb or olfactory subarachnoid space following intranasal administration: (1) receptor-mediated endocytosis into olfactory sensory neurons followed by slow intracellular transport (from hours to days) to the olfactory bulb, (2) non-specific endocytosis into olfactory sensory neurons followed by intracellular transport to the olfactory bulb, and (3) extracellular diffusion into the olfactory submucosa along open intercellular clefts in the olfactory epithelium with a rapid transport (∼30 min) directly to the olfactory bulb or the olfactory subarachnoid space and entrance to the cerebrospinal fluid (CSF) circulation. IND-1233-ASO could be rapidly transported to the brain following intranasal administration mediated by the pulsatile flow through CSF leading to subsequent uptake into monoaminergic neurons via functional monoamine transporters. CPu, caudate putamen; DR, dorsal raphe nucleus; LC, locus coeruleus; SNc/VTA, substantia nigra compacta/ventral tegmental area.
Figure 3
Figure 3
Intranasal Indatraline-Conjugated 499-siRNA Treatment Downregulates α-Synuclein Expression (A) Schematic representation of the treatment. Mice were intranasally administered with PBS, indatraline-conjugated nonsense siRNA (IND-NS-siRNA), or indatraline-conjugated 499-siRNA (IND-499-siRNA) at 30 μg/day for 4 days and were sacrificed at 1, 3, or 7 days after last administration (1, 3, or 7 days, respectively; n = 5–10 mice/group). (B) Coronal brain sections showing reduced α-synuclein mRNA levels in the SNc, VTA, DR, and LC of mice treated with IND-499-siRNA (4 days) and sacrificed at 1 day post-administration were assessed by in situ hybridization (ISH). Signal represents the relative optical density (ROD) of autoradiograms as indicated at the right-hand side of the image. White arrowheads show α-synuclein mRNA expression in SNc, VTA, DR and LC. Scale bar: 500 μm. (C) Time course of α-synuclein mRNA suppression in the monoaminergic nuclei after intranasal IND-499-siRNA administration. Bar graphs showing a significant reduction of α-synuclein mRNA level compared with their respective controls at day 1 post-administration. Conversely, no difference was detected at days 3 and 7 post-administration. **p < 0.01, ***p < 0.001, versus PBS-treated mice; +p < 0.05, ++p < 0.01, +++p < 0.001 versus IND-NS-siRNA (two-way ANOVA followed by Tukey’s post hoc test). (D) Image of immunoblot of α-synuclein, β-actin, and tyrosine hydroxylase (TH) in SNc/VTA of mice treated with PBS or IND-conjugated siRNAs. (E) Bar graphs showing α-synuclein protein levels in SNc/VTA normalized against β-actin or TH, and TH protein levels normalized against β-actin. IND-499-siRNA reduced α-synuclein protein level in SNc/VTA 24 hr after last administration; then α-synuclein level was recovered 3 days later. *p < 0.05 versus control groups (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. DR, dorsal raphe nucleus; LC, locus coeruleus; SNc/VTA, substantia nigra compacta/ventral tegmental area.
Figure 4
Figure 4
Intranasal Indatraline-Conjugated 1233-ASO Treatment Downregulates α-Synuclein Expression (A) Schematic representation of the treatment. Mice were intranasally administered with PBS, indatraline-conjugated nonsense ASO (IND-1227-ASO), or indatraline-conjugated 1233-ASO (IND-1233-ASO) at 30 μg/day for 4 days and were sacrificed at 1, 3, or 7 days after last administration (1, 3, or 7 days, respectively; n = 5–10 mice/group). (B) Coronal brain sections showing reduced α-synuclein mRNA levels in the SNc, VTA, DR, and LC of mice treated with IND-1233-ASO (4 days) and sacrificed at 1 day post-administration assessed by in situ hybridization (ISH). Signal represents the relative optical density (ROD) of autoradiograms as indicated at the right-hand side of the image. White arrowheads show α-synuclein mRNA expression in SNc, VTA, DR and LC. Scale bar: 500 μm. (C) Time course of α-synuclein mRNA suppression in the monoaminergic nuclei after intranasal IND-1233-ASO administration. Bar graphs showing a significant reduction of α-synuclein mRNA level compared with their respective controls at day 1, but not at 3 and 7 days post-administration. *p < 0.05, **p < 0.01 versus PBS-treated mice; +p < 0.05, ++p < 0.01, +++p < 0.001 versus IND-1227-ASO (two-way ANOVA followed by Tukey’s post hoc test). (D) Image of immunoblot of α-synuclein, β-actin, and tyrosine hydroxylase (TH) in SNc/VTA of mice treated with PBS or IND-conjugated ASOs. (E) Bar graphs showing α-synuclein protein levels in SNc/VTA normalized against β-actin or TH, and TH protein levels normalized against β-actin. IND-1233-ASO decreased α-synuclein protein levels in SNc/VTA up to 3 days post-treatment, with a subsequent recovery to basal values at 7 days. *p < 0.05 versus control groups (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. DR, dorsal raphe nucleus; LC, locus coeruleus; SNc/VTA, substantia nigra compacta/ventral tegmental area.
Figure 5
Figure 5
Neurochemical Effects on Forebrain DA Neurotransmission of IND-1233-ASO-Induced α-Synuclein Knockdown Mice were intranasally administered with PBS, indatraline-conjugated nonsense ASO (IND-1227-ASO), or indatraline-conjugated 1233-ASO (IND-1233-ASO) at 30 μg/day for 4 days. Microdialysis experiments were conducted between 1 and 3 days after last administration. (A and F) Local veratridine infusion (depolarizing agent, 50 μM) by reverse dialysis increased extracellular DA release in CPu (A) and mPFC (F). This effect was more significant in IND-1233-ASO-treated mice than in control groups. (B and G) Nomifensine (DAT inhibitor, 1-10-50 μM) increased more significantly extracellular DA levels in CPu (B) and mPFC (G) of α-synuclein knockdown mice than in control groups. (C and H) Similarly, local infusion of amphetamine (DA releaser, 1-10-100 μM) induced a higher DA release in CPu (C) and mPFC (H) of IND-1233-ASO-treated mice versus control mice. (D and I) Local tetrabenazine (VMAT2 inhibitor, 100 μM) application significantly reduced DA release in both brain areas: CPu (D) and mPFC (I). Only in the CPu (D) was this effect faster in control mice than in α-synuclein knockdown mice. (E and J) Local activation of DA D2/3 receptor in CPu (E) and mPFC (J) by the infusion of 10 μM quinpirole by reverse dialysis reduced DA release in all treatments. However, after quinpirole was removed, control groups recovered baseline DA levels faster than α-synuclein knockdown mice. The number of mice used in each experiment is shown in parentheses. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control groups (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. CPu, caudate putamen; mPFC, medial prefrontal cortex.
Figure 6
Figure 6
Neurochemical Effects on Forebrain 5-HT Neurotransmission of IND-1233-ASO-Induced α-Synuclein Knockdown Mice were intranasally administered with PBS or indatraline-conjugated 1233-ASO (IND-1233-ASO) at 30 μg/day for 4 days. Microdialysis experiments were conducted between 1 and 3 days after last administration. (A and F) Local veratridine infusion (depolarizing agent, 50 μM) by reverse dialysis increased extracellular 5-HT release in CPu (A), but not in mPFC (F). This effect was more significant in IND-1233-ASO-treated mice than in PBS-treated mice. (B and G) Citalopram (SERT inhibitor, 1-10-50 μM) increased more significantly extracellular 5-HT levels in CPu (B) and mPFC (G) of α-synuclein knockdown mice than in PBS mice. (C and H) Local tetrabenazine (VMAT2 inhibitor, 100 μM) application significantly reduced 5-HT release in CPu (C) and mPFC (H). However, only CPu reached a marginal statistical difference between phenotypes (p = 0.053), with a greater effect of tetrabenazine in control than in α-synuclein knockdown mice. (D and I) Local activation of 5-HT1B receptor in CPu (D) and mPFC (I) by the infusion of 300 μM CP93129 by reverse dialysis reduced 5-HT release in PBS and α-synuclein knockdown mice. However, after CP93129 was removed, PBS mice recovered baseline 5-HT levels more rapidly than α-synuclein knockdown mice. (E and J) Systemic 8-OH-DPAT (5-HT1A receptor agonist, 1 mg/kg i.p.) administration reduced 5-HT release similarly in CPu (E) and mPFC (J) of PBS and α-synuclein knockdown mice. The number of mice used in each experiment is shown in parentheses. *p < 0.05, **p < 0.01, ***p < 0.001 compared with PBS mice (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. CPu, caudate putamen; mPFC, medial prefrontal cortex.
Figure 7
Figure 7
Neurochemical Effects on Nigrostriatal DA Neurotransmission in Transgenic Mice Overexpressing α-Synuclein Transgenic mice expressing human wild-type α-synuclein cDNA under the control of TH promotor (TG+) and their respective controls (TG) were implanted with a microdialysis probe into CPu. Microdialysis experiments were conducted between 1 and 3 days post-implantation. (A) Local veratridine infusion (depolarizing agent, 50 μM) by reverse dialysis increased extracellular DA release in CPu, but this effect was more significant in TG- mice than in TG+ mice. (B) Local nomifensine (DAT inhibitor, 1-10-50 μM) application increased more significantly extracellular DA levels in CPu of TG mice than in TG+ mice. (C) However, local infusion of amphetamine (DA releaser, 1-10-100 μM) increased striatal DA levels similarly in both phenotypes. (D) In addition, tetrabenazine (VMAT2 inhibitor, 100 μM) induced a comparable effect on striatal DA release in TG and TG+ mice. (E) Local activation of DA D2/3 receptor in CPu by the infusion of 10 μM quinpirole by reverse dialysis only reduced DA release in TG mice, but not in TG+ mice. The number of mice used in each experiment is shown in parentheses. *p < 0.05, ***p < 0.001, compared with TG mice (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. CPu, caudate putamen.

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