siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain

Abstract

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNAinduced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain. The serotonin transporter (SERT) controls the temporal and spatial activity of extracellular serotonin by facilitating a rapid and high-affinity reuptake of this neurotransmitter into presynpatic terminals. 1 SERT is also a primary molecular target for the most widely prescribed antidepressant drugs, the selective serotonin reuptake inhibitors (SSRIs). 2 SSRIs readily inhibit SERT activity and elevate the serotonergic tone in the brain. However, full therapeutic benefits ensue only after chronic use of SSRIs, requiring the manifestation of long-term adaptations, secondary to the blockade of serotonin reuptake. 3,4 Emerging studies reveal downregulation of SERT as an adaptive consequence of sustained and high occupancy by chronically administered SSRIs. 5-8 The downregulation of SERT favorably potentiates the SSRI-mediated increase in the brain serotonergic neurotransmission. [6][7] Further, the lag time required by SSRIs to produce a substantial downregulation of SERT correlates well with the time needed for eliciting a full therapeutic response. Therefore, downregulation of SERT has been implicated in the antidepressant action of SSRIs. 7 Recently, SERT-deficient mice were used ...

Click-Modified Cyclodextrins as Nonviral Vectors for Neuronal siRNA Delivery

“…45−47 For example, a 30% decrease in striatal tyrosine hydroxylase was sufficient to reduce the hyperactive locomotor response to amphetamine in mice 46 and antidepressant-like behaviors were observed in mice, after icv infusion of siRNA led to a 40% reduction in expression of the serotonin transporter. 47 In summary, an amphiphilic cationic modified β-cyclodextrin was shown to deliver siRNA to neurons for gene silencing. Efficient uptake into neuronal cells was shown, with a favorable cytotoxicity profile.…”

Evidence for significant contribution of a newly identified monoamine transporter (PMAT) to serotonin uptake in the human brain

“…The mRNA-induced 5-HT uptake was measured in transport assays, and the effect of PMAT-specific antisense oligos was investigated. To ensure specificity, we included sense oligos as controls and designed both PMAT and SERT oligos based on sequences that were shown to be effective and specific in previous RNAi studies [12,22]. Our results showed that pre-hybridization of total human brain poly(A) + mRNA with PMAT antisense substantially reduced mRNA-induced 5-HT uptake, suggesting that functional PMAT transcripts are present in the human brain, and PMAT may be significantly involved in total brain uptake of 5-HT.…”

“…At clinically encountered concentrations in the brain (low nanomolar range) (Table 3) Illustration of positions of PMAT and SERT oligos used in hybrid depletion study. * values are calculated as steady state plasma concentrations of total drug times plasma unbound fraction using data from [22,23], assuming that unbound brain concentrations of SSRIs approximate their unbound plasma concentrations [32].…”

“…These oligonucleotides (oligos) demonstrated high specificity towards PMAT when blasted using the human genome database. For SERT, two pairs of nucleotides (18-21 nt) were designed based on the RNAi study of Thakker et al, where they were shown to be effective in blocking SERT expression [22]. The locations and sequences of oligos used in this study were shown in Figure 1 and Table 1.…”

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

“…Upon local multidosal administration, naked unmodified siRNA silences target gene expression in the lung, the eye or the central nervous system. [4][5][6] Systemic applications of RNAi, in contrast, are hampered by rapid degradation and renal excretion. 2 To improve systemic delivery, siRNA delivery vehicles based on conjugates, liposomes or nanoparticles are being developed.…”