Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration

Abstract

Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.

Figure 1

Selective accumulation of sertraline-conjugated nonsense-siRNA (C-NS-siRNA) in tryptophan hydroxylase2-positive (TPH₂-positive) 5-hydroxytryptamine (5-HT) neurons after intranasal administration. Mice were intranasally administered with alexa488 phosphate-buffered saline (PBS) (A488-PBS) or alexa488-labeled C-NS-siRNA (A488-C-NS-siRNA) at 30 μg day⁻¹ during 4 days and were killed 6 h postadministration (n=2 mice/group). (a) Confocal images showing co-localization of A488-C-NS-siRNA (yellow) in dorsal raphe nucleus (DR) 5-HT neurons (TPH₂-positive, red) identified with white arrowheads. Cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole; blue). Bottom row are high-magnification photomicrographs of the frames in top row. Scale bars: low=200 μm, high=10 μm. (b and c) Histograms show the distribution profile of the abundance of A488-C-NS-siRNA (expressed as fluorescence units, ranges shown below the abscissa axis) in TPH₂-positive neurons. Note the greater number of TPH₂-positive cells co-localized with A488-C-NS-siRNA in the DR compared with median raphe nucleus (MnR). Range: >3, 3–1 and <1 represent relative unit of intracellular A488 density. (d) Number of TPH₂-positive cells in the DR and MnR of mice. AP coordinates (in mm): −4.24/−4.36 and −4.48/−4.72 from bregma (n=2 mice/group). *P<0.05, **P<0.01, ***P<0.001 versus A488-PBS-treated mice. Data are mean±s.e.m.

Figure 2

Intranasal sertraline-conjugated serotonin transporter small interfering RNA (SERT-siRNA) (C-SERT-siRNA) treatment downregulates SERT expression. Mice received intranasally: phosphate-buffered saline (PBS), sertraline-conjugated nonsense-siRNA (C-NS-siRNA) or C-SERT-siRNA at 30 μg day⁻¹ (2.1 nmol day⁻¹) during 1, 4 or 7 days. (a) Coronal brain sections showing reduced SERT mRNA and binding site levels in the dorsal raphe nucleus (DR) (AP coordinates: −4.48 to −4.72 in mm) of mice treated with C-SERT-siRNA (7-day). Scale bar: 500 μm. (b) Effects of C-SERT-siRNA on SERT mRNA and binding site densities in the DR and median raphe nucleus (MnR) (n=3–8 mice/group; *P<0.05, **P<0.01, ***P<0.001 versus PBS- and C-NS-siRNA-treated mice). (c) Immunohistochemistry images showing the expression of SERT protein (SERT-ir) in mouse DR. Bottom row are high-magnification photomicrographs of the frames in top row. Scale bars: low=100 μm, high=20 μm. (d) C-SERT-siRNA treatment (7-day) decreased DR SERT protein density versus PBS- and C-NS-siRNA-treated mice (n=3–5 mice/group; **P<0.01). (e) Local selective serotonin reuptake inhibitor citalopram infusion by reverse-dialysis induced concentration-dependent increases of extracellular 5-hydroxytryptamine (5-HT) in the caudate putamen (CPu) of PBS-treated mice more than in C-SERT-siRNA-treated mice (n=7–8 mice/group; **P<0.01 versus PBS). Data are mean±s.e.m.

Figure 3

RNA interference-induced serotonin transporter (SERT) suppression reduces 5-hydroxytryptamine 1A (5-HT_1A)-autoreceptor expression/function and rapidly enhances the forebrain 5-HT transmission. Mice were intranasally administered with: phosphate-buffered saline (PBS), sertraline-conjugated nonsense-small interfering RNA (siRNA) (C-NS-siRNA) or sertraline-conjugated SERT-siRNA (C-SERT-siRNA) at 30 μg day⁻¹ during 7-day treatment. Other groups of mice were treated with saline or fluoxetine (FLX) at 10 mg kg⁻¹ day⁻¹, intraperitoneally during 7- or 28-day treatment. (a) Representative coronal brain sections showing reduced 5-HT_1A receptor mRNA and binding site levels in the dorsal raphe nucleus (DR) of C-SERT-siRNA-treated mice. Scale bar: 2 mm. (b) Effects of C-SERT-siRNA on 5-HT_1A receptor mRNA and binding site densities in the DR and median raphe nucleus (MnR) of mice (n=3–4 mice/group; *P<0.05, **P<0.01 compared with PBS- and C-NS-siRNA-treated mice). (c) Effects of C-SERT-siRNA and FLX on 5-HT_1A-autoreceptor function. C-SERT-siRNA (7-day) decreased 5-HT_1A receptor-mediated 8-OH-DPAT-stimulated [³⁵S]GTPγS binding in the DR, whereas FLX (7-day) was without effect (n=3–4 mice/group; **P<0.01 versus PBS- and C-NS-siRNA-treated mice). FLX reduced 5-HT_1A-autoreceptor function after 28-day treatment (n=3–4 mice/group; ^τττP<0.001 versus saline and FLX 7-day). (d) 8-OH-DPAT did not reduce 5-HT release in the ventral hippocampus (HPCv) of C-SERT-siRNA-treated mice (7-day), unlike control groups (n=3–4 mice/group; **P<0.01 versus PBS and C-NS-siRNA). However, 8-OH-DPAT decreased hippocampal 5-HT concentration in saline- and FLX-treated 7-day mice but not in 28-day FLX-treated mice (n=5–8 mice/group; ^ττP<0.01 versus saline and FLX 7-day). (e) Intranasal C-SERT-siRNA treatment increased extracellular 5-HT levels in CPu more rapidly than FLX (n=4–10 mice/group; **P<0.01, ***P<0.001 versus PBS and C-NS-siRNA; ^τP<0.05, ^ττP<0.01, ^τττP<0.001 versus saline). Significant differences versus their respective control mice occurred after 2-day C-SERT-siRNA and after 7-day FLX treatment. Similar temporal differences were observed in HPCv (n=3-6 mice/group; ***P<0.001 versus PBS and C-NS-siRNA; ^τP<0.05, ^τττP<0.001 versus saline). Data are mean±s.e.m.

Figure 4

Intranasal administration of sertraline-conjugated serotonin transporter small interfering RNA (SERT-siRNA) (C-SERT-siRNA) accelerates the proliferation of cellular precursors and dendrite complexity in the hippocampus. (a) Representative images showing an increased number of Ki67-positive cells in the dentate gyrus (DG) of C-SERT-siRNA (7-day) or fluoxetine-treated mice (FLX, 28-day) versus their respective control mice. Bottom row shows high-magnification photomicrographs of the top row frames. Scale bars: low=100 μm and high=20 μm. (b and c) Short-term C-SERT-siRNA (7-day) or long-term FLX (28-day) treatments increased similarly the number of DG Ki-67-positive cells (n=5–10 mice/group) and 5-bromo-2'-deoxyuridine (BrdU)-positive cells (n=6–10 mice/group). *P<0.05, **P<0.01 versus phosphate-buffered saline (PBS) and sertaline-conjugated nonsense-siRNA (C-NS-siRNA); ^τP<0.05 versus saline and FLX 7-day treatment. (d and e) Short-term C-SERT-siRNA treatment or chronic FLX administration increased similarly the number of immature neurons identified with NeuroD (n=4–10 mice/group) or doublecortin (DCX; n=5-11 mice/group) markers (*P<0.05 versus PBS and C-NS-siRNA; ^τP<0.05, ^ττP<0.01 versus saline and FLX 7-day treatment). (f) Representative images and traces from Sholl analyses of DCX-positive cells bearing a complex dendritic morphology in the DG of mice in the different treatment group. Scale bar: 20 μm. (g) Effects of C-SERT-siRNA (7-day) or FLX (28-day) treatments on dendritic intersection numbers and dendritic length of DCX-positive neurons (n=4 mice/group, 5 cells/mouse; ***P<0.001 versus PBS and C-NS-siRNA; ^τττP<0.001 versus saline and FLX 7-day). (h) Levels of mRNA for the following genes: BDNF, VEGF, TRKB, ARC, Neuritin, and PSD95 in the DG were analyzed by densitometry and are shown in the bar graphs (n=3–10 mice/group; *P<0.05, ***P<0.001 versus PBS and C-NS-siRNA; ^τP<0.05, ^ττP<0.01, ^τττP<0.001 versus saline and FLX 7-day treatment). Values are mean±s.e.m.

Figure 5

Short-term intranasal (i.n.) treatment with sertraline-conjugated serotonin transporter small interfering RNA (SERT-siRNA) (C-SERT-siRNA) efficiently attenuates the behavioral deficits in a stress-induced depression model. Grouped-housed male C57BL/6J mice were presented during 28 or 49 days with vehicle (non-stressed mice) or corticosterone (stressed mice) in the presence or absence of an antidepressant treatment (C-SERT-siRNA 30 μg day⁻¹, i.n. or fluoxetine (FLX) 10 mg kg⁻¹ day⁻¹, intraperitoneally) during the last 7 or 28 days of the corticosterone regimen. (a and b) Short-term C-SERT-siRNA reversed the reduction of sucrose intake and preference in the corticosterone-induced anhedonia (n=10–16 mice/group; ***P<0.001 versus non-stressed mice; ^^^P<0.001 versus corticosterone-stressed mice treated with phosphate-buffered saline (PBS) or sertraline-conjugated nonsense-siRNA (C-NS-siRNA)). FLX induced a similar recovery after 28-day, but not after 7-day, treatment (n=7–12 mice/group; ^τττP <0.001 versus non-stressed mice; ^^^P<0.001 versus corticosterone-stressed mice treated with saline. (c) Effect on novelty suppressed feeding test (NSFT). Seven-day C-SERT-siRNA, but not 7-day FLX, reversed the increased latency to feed in corticosterone-treated mice (n=8–12 mice/group; **P<0.01 versus non-stressed mice; ^P<0.05 versus corticosterone-stressed mice treated with PBS or C-NS-siRNA). Similar effects were elicited by 28-day FLX administration (n=7–12 mice/group; ^ττP<0.01 versus non-stressed mice; ^P<0.05 versus corticosterone-stressed mice treated with saline). (d) Survival analysis of NSFT data. (e) C-SERT-siRNA (7-day) or FLX (28-day), but not FLX 7-day, decreased the immobility time in the tail suspension test (TST) in cortico-stressed mice (n=8–15 mice/group). **P<0.01, ***P<0.001, ^τP<0.05, ^ττP<0.01 versus their non-stressed mice, respectively; ^P<0.05, ^^P<0.01 versus cortico-stressed mice treated with saline, PBS or C-NS-siRNA, respectively. Values are mean±s.e.m.