Serotonin (5-HT) plays important roles in the central nervous system (CNS), participating in control of various motor, physiological, affective, and cognitive functions. Disturbances in serotonergic neurotransmission are involved in several neuronal and psychiatric diseases, including Alzheimer's disease, anxiety, and, in particular, major depression.
Among the proteins implicated in 5-HT neurotransmission, the serotonin transporter (SERT) is a key component, regulating both intensity and duration of the effects of 5-HT released into the synaptic cleft. Numerous psychoactive drugs target the SERT, including antidepressant drugs, such as selective serotonin reuptake inhibitors (SSRIs), which increase extracellular 5-HT (Thomas et al., 1987; Dechant and Clissold, 1991) and some drugs of abuse, such as cocaine (Lesch, 1997) and the amphetamine-derivative 3,4-meth-ylenedioxymetamphetamine (MDMA or "Ecstasy") (Green et al., 1995). The contribution of SERT to 5-HT neurotransmission is limited by the lack of pharmacological approaches for modulating SERT levels in the CNS. In this context, the cloning of the SERT gene in different species (Blakely et al., 1991; Lesch et al., 1993; Chang et al., 1996) raised new opportunities for modifying SERT expression.
The most drastic approach was the creation of mice lacking the SERT (Bengel et al., 1998). However, the resulting profound alterations of the 5-HT systems (Bengel et al., 1998; Rioux et al., 1999; Fabre et al., 2000a; Mannoury La Cour et al., 2001), beginning probably from embryonic stages, are not relevant to antidepressant treatment in the adult and alternative approaches are needed.
Antisense oligodeoxynucleotide (ODN) administration can specifically inhibit expression of targeted proteins; for example, rat dopamine transporter (DAT; Silvia et al., 1997). However, major drawbacks include low stability, toxicity, and weak penetration of ODNs into cells. Their short action is particularly limiting when the target protein has a long half-life, as is the case for DAT (Silvia et al., 1997; Martres et al. 1998), or
Transmembrane Transporters. Edited by Michael Quick
Copyright © 2002 by Wiley-Liss, Inc.
ISBNs: 0-471-06517 (Hardcover); 0-471-43404-3 (Electronic)
when long-lasting effects are necessary to study a phenomenon such as antidepressant effects.
An alternative method is direct gene transfer into the CNS (Fig. 6.1). Various strategies have been used: grafting of genetically modified cells (Jiao et al., 1993; Sabate et al., 1995; Bachoud-Levi et al., 2000) or direct in vivo gene delivery by viral vectors, including adenoviruses (Akli et al., 1993; Horellou et al., 1994), herpes-virus derived vectors (During et al., 1994; Kesari et al., 1995; Antunes Bras et al., 1998), and adeno-associated viruses (Kaplitt et al., 1994). However, cell- and viral-mediated gene transfer procedures raise safety considerations as to the possible immunogenic and/or pathogenic effects of the vectors used (see Fisher and Ray, 1994; Crystal, 1995, 1996; Danos, 2000, for reviews).
In this respect, nonviral techniques offer numerous advantages. Chemical DNA carriers are of greater flexibility in use, and the risk of secondary complications is reduced (Crystal, 1995, 1996). Two main classes of molecules can be distinguished. Cationic lipids have been developed (Weiss et al., 1997; Byk et al., 1998), but amounts of DNA encapsulated are low and the efficacy of gene transfer is limited.
Cationic polymers display several advantages, including lesser toxicity and the possibility of transferring DNA of much larger size (see Lemkine and Demeneix, 2001, for review). Both the absence of toxicity of polyethylenimine (PEI) and its efficiency for transferring DNA in the adult rodent brain (Boussif et al., 1995; Abdallah et al., 1996; Goula et al., 1998; Martres et al., 1998) prompted us to use this approach to deliver sense or antisense SERT DNA coding sequences directly into the dorsal raphe nucleus (DRN) of adult rats (Fabre et al., 2000b). The resulting changes in SERT expression were assessed and possible functional alterations in 5-HT neurotransmission were followed by measuring 5-HT turnover and analyzing the binding and functional properties of the 5-HT1Areceptors and recording the sleep-wakefulness rhythm.
All experiments were performed in conformity with institutional guidelines in compliance with national and international law and policies for use of animals in neuroscience research. Rats were housed under standard laboratory conditions: 12:12 h light-dark cycle (light on at 07:00), 22 ± 1°C ambient temperature, 60% relative humidity, food and water ad libitum.
Construction of Recombinant Plasmids and Preparation of DNA/PEI Complexes
For construction of the SERT sense plasmid, a cDNA comprising the entire coding sequence of the rat SERT (nucleotides 87 to 2400; Blakely et al., 1991) was subcloned into the pRc-CMV expression vector (Invitrogen, Cezgy Pontoise, France), containing the cytomegalovirus promoter and the bovine growth hormone (BGH) polyadenylation signal. For the antisense constructs, the complete coding sequence of SERT ("full antisense") or its last 468 nucleotides (nucleotides 1540-2007, "short antisense") were subcloned in reverse orientation into pRc-CMV (Fig. 6.2A). Plasmid DNAs were prepared by two cen-trifugations in CsCl gradient, resuspended in 10 mM Tris-HCl and 1 mM ethylene diamine tetraacetate (EDTA), pH 8, quantified (260 nm O.D.) and stored at -20°C.
The PEI concentration necessary to neutralize the DNA anionic charges was determined as follows: 0.5 pg of the various DNA preparations were mixed with PEI at different cationic charge equivalents (calculated by taking into account that 1 pg of DNA and 1 pl of 0.1 M PEI correspond, respectively, to 3 nmoles of phosphate and 100 nmoles of amine nitrogen). The DNA/PEI complexes were then electrophoresed in a 1% agarose gel and their migration profiles were compared.
Measurement of [3H]5-HT Uptake in Transfected Cells
LLC-PK1 cells (pig kidney epithelial cells, ATCC n: CRL1392) were grown under a 7% O2/93% air atmosphere at 37°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin G, and 10 mg/ml streptomycin. DNA constructs were transfected into 60-80% confluent cells by electroporation (3-5 x 106 cells in 500 pl of DMEM without serum, 270 V, 1800 pF, relaxation time: 40 ms). Twenty-four hours after transfection, cells were transferred into 24-well plates and 24 h later expression of SERT measured by [3H]5-HT uptake (Martres et al.; 1998). Cells were washed twice with 1 ml of uptake buffer (4 mM Tris, 6.25 mM N-(2-hydroxyethyl) piperazine-2'-(2-ethanesulphonic acid) (HEPES), 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 5.6 mM glucose, and 0.5 mM ascorbic acid, final pH 7.4) and incubated in 500 pl of uptake buffer containing 3-6 nM [3H]5-HT (15 Ci/mmol, Amersham, Buckinghamshire, UK), without or with 10 pM fluoxetine (Lilly, Indianapolis, IN, USA) to determine nonspecific uptake. After 7 min at 37°C, cells were washed quickly three times with 1 ml of uptake buffer at room temperature (RT), then solubilized in 500 pl of 0.1 N NaOH and the radioactivity counted by liquid scintillation spectrometry.
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