Dieter Hüsken

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

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 siRNA-induced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain.

mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides

BACKGROUND A general method allowing the selective destruction of targeted mRNA molecules in vivo would have broad application in biology and medicine. Metal complexes are among the best synthetic catalysts for the cleavage of RNA, and covalent attachment of suitable metal complexes to oligonucleotides allows the cleavage of complementary single-stranded RNAs in a sequence-specific manner. RESULTS Using novel europium complexes covalently linked to an oligodeoxyribonucleotide, we have achieved the sequence-specific cleavage of a complementary synthetic RNA. The complexes are completely resistant to chemical degradation under the experimental conditions. The cleavage efficiency of the conjugate strongly depends on the nature of the linker between the oligonucleotide and the complex. Almost complete cleavage of the RNA target has been achieved within 16 h at 37 degrees C. CONCLUSIONS The results will be important for improving the efficacy of antisense oligonucleotides and will provide a basis for the design of synthetic RNA restriction enzymes. Conjugates of the kind described here may also find application as chemical probes for structural and functional studies of RNA.

Development of artificial ribonucleases

Antisense oligonucleotides offer a new way of treating genetically based diseases by interfering with protein expression on the mRNA level. First generation phosphorothioate antisense oligonucleotides have shown promising results in animal experiments and several compounds are currently being tested in the clinics against a variety of diseases. Nevertheless, improvements of various aspects of the technology, such as stability, reduction of length and target affinity of oligonucleotides, are still highly desirable. As a consequence, a large number of chemical modifications of antisense compounds has been reported during the last decade. Unlike phosphorothioates, however, most second generation modifications do not induce degradation of messenger RNA by an RNase H-based mechanism, which is in most cases an essential component of antisense activity. An alternative approach to enable degradation of the mRNA comprises the covalent attachment of RNA cleaving groups to modified oligonucleotides. Macrocyclic lanthanide complexes, in combination with modified antisense oligonucleotides, can be used to specifically cleave mRNA. The preparation, properties and use of such artificial ribonucleases are highlighted. In particular, the design and preparation of constructs cleaving RNA with multiple turn-over is described.

Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented.

SEQUENCE-SPECIFIC CLEAVAGE OF RNA USING MACROCYCLIC LANTHANIDE COMPLEXES CONJUGATED TO OLIGONUCLEOTIDES : A STRUCTURE ACTIVITY STUDY

Abstract A series of lanthanide complexes were synthesized and conjugated to an oligodeoxynucleotide. Sequence-specific cleavage of a complementary RNA by the conjugates was performed and results are discussed in terms of structure of the complexes.

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids.

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA.

Duplex- and triplex-forming properties of 4'-thio-modified oligodeoxynucleotides

Abstract The melting temperatures (Tms) of a series of 4′-thio-modified oligodeoxynucleotides bound to complementary ssDNA, ssRNA and dsDNA have been determined. The results demonstrate an increase in Tm for the duplexes formed with RNA, but a reduction when bound to DNA. When forming a triplex with dsDNA, the greatest increases in Tm are observed when the 4′-thio-modifications are placed in contiguous stretches.

Novel polyamide based nucleic acid analogs-synthesis of oligomers and RNA-binding properties

Abstract The synthesis of novel polyamide based nucleic acid analogs incorporating monomer units of type I – III has been accomplished using solid-phase strategies based on N δ -Fmoc protected building blocks. An oligomer composed of monomer units I exhibited weak, but sequence-specific RNA binding. Improved RNA-binding affinity was observed for analogs incorporating building blocks of type III with 2-(R) stereochemistry, but not in the case of the 2-(S) isomers.

New approaches towards fluorescence labelling of messenger RNA transcripts.

Fluorescence labelling of nucleic acids is being used for a wide range of biological applications. The performance of these techniques is dependent on fluorochrome labels with a high sensitivity and high resistance to photobleaching. Indo-cyanine dyes such as Cy3, Cy5 or Cy7 have been found to fulfil these requirements. This study describes several different RNA labelling techniques allowing for a Cy5 based detection of mRNA transcripts.